Enzymatic systems for carbon fixation and methods of generating same

ABSTRACT

A system for carbon fixation is provided. The system comprises enzymes which catalyze reactions of a carbon fixation pathway, wherein at least one of the reactions of the carbon fixation pathway is a carboxylation reaction, wherein products of the reactions of the carbon fixation pathway comprise oxaloacetate and malonyl-CoA, wherein an enzyme which performs the carboxylation reaction is selected from the group consisting of phophoenolpyruvate (PEP) carboxlase, pyruvate carboxylase and acetyl-CoA carboxylase and wherein an export product of the carbon fixation pathway is glyoxylate. Additional carbon fixation pathways are also provided and methods of generating same.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/576,720 filed on Aug. 2, 2012, which is a National Phase of PCTPatent Application No. PCT/IL2011/000145 having International FilingDate of Feb. 10, 2011, which claims the benefit of priority of U.S.Provisional Patent Application No. 61/303,338 filed on Feb. 11, 2010.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to enzymatic systems for carbon fixationand methods of generating same.

Photosynthesis is a process executed by photosynthetic organisms bywhich, inorganic carbon (Ci), such as CO₂ and HCO₃, is incorporated intoorganic compounds using the energy of photon radiation. Photosyntheticorganisms, such as, soil-grown and aquatic plants and cyanobacteria(blue-green algae), depend on the organic compounds produced viaphotosynthesis for sustenance and growth.

In the process of transforming sunlight into biological matter, plantsabsorb ten times more carbon dioxide from the atmosphere than is emittedby the global human population. Moreover, agriculture, which isdependent on carbon fixation, consumes over 70% of the fresh waterutilized by humanity and the majority of cultivatable land resources onearth. These figures point to the central place that carbon fixation byplants plays in our global ecological footprint. In nature the growthlimiting factors of photosynthetic organisms vary between habitats andoften include the availability of water, light, fixed nitrogen, iron andphosphorous. However, under human cultivation the usage of fertilizersand irrigation can make the carbon fixation rate limiting; for example,various C3 plants have shown a significant increase in growth rate whenexposed to twice the atmospheric CO₂ concentration.

Previous growth enhancements have been demonstrated by addressingseveral biochemical limiting factors, related both to thelight-dependent and light-independent reactions. For example, transgenicArabidopsis plants that expressed an efficient bacterialphotorespiration pathway, instead of their natural photorespirationpathway, grew faster, produced more shoot and root biomass, andcontained more soluble sugars [Kebeish R, et al. (2007) Chloroplasticphotorespiratory bypass increases photosynthesis and biomass productionin Arabidopsis thaliana. Nat Biotechnol 25(5):593-599]. In anothereffort, tobacco plants overexpressing sedoheptulose-1,7-bisphosphatase,an enzyme operating in the reductive pentose phosphate cycle (rPP, alsoknown as the Calvin-Benson Cycle), were characterized by an increasedphotosynthetic rate and a 30% enhancement in biomass yield [Lefebvre S,et al. (2005) Increased sedoheptulose-1,7-bisphosphatase activity intransgenic tobacco plants stimulates photosynthesis and growth from anearly stage in development. Plant Physiol 138(1):451-460].

The rPP cycle (FIG. 5A), used by the vast majority of autotrophicorganisms for CO₂ assimilation, is limited by the slow rate of Rubisco(Ribulose-1,5-bisphosphate carboxylase/oxygenase). The inversecorrelation between the enzyme turnover number (˜2-4 s⁻¹) and its CO₂specificity indicates that the enzyme might already be naturallyoptimized. Therefore, further optimization of Rubisco may provedifficult and lead to only marginal results [Raines C A (2006)Transgenic approaches to manipulate the environmental responses of theC3 carbon fixation cycle. Plant Cell Environ 29(3):331-339] therebylimiting the potential for increasing the rate of the rPP cycle.Designing and developing alternative (Rubisco independent) pathways thatcan support carbon fixation with a higher rate can therefore be highlybeneficial.

To date, five natural metabolic pathways have been identified that arecapable of performing carbon fixation in place of the classic rPP cycle.These are the reductive tri-carboxylic-acid (rTCA) cycle, postulated inthe 60's; the oxygen sensitive reductive acetyl-CoA (rAcCoA) pathway;the extensively researched 3-hydroxypropionate (3-HP) cycle; the3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle and the recentlydiscovered dicarboxylate/4-hydroxybutyrate (DC/4-HB) cycle.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a system for carbon fixation, comprising enzymes whichcatalyze reactions of a carbon fixation pathway, wherein at least one ofthe reactions of the carbon fixation pathway is a carboxylationreaction, wherein products of the reactions of the carbon fixationpathway comprise oxaloacetate and malonyl-CoA, wherein an enzyme whichperforms the carboxylation reaction is selected from the groupconsisting of phophoenolpyruvate (PEP) carboxylase, pyruvate carboxylaseand acetyl-CoA carboxylase and wherein an additional product of thecarbon fixation pathway is glyoxylate.

According to some embodiments of the invention, the glyoxylate is theexport product.

According to some embodiments of the invention, pyruvate is the exportproduct.

According to some embodiments of the invention, the enzymes of thecarbon fixation pathway generate more than 0.3 μmolglyceraldehyde-3-phosphate/min/mg.

According to some embodiments of the invention, the enzyme whichperforms the carboxylation enzyme is PEP carboxylase.

According to some embodiments of the invention, at least two of thereactions of the carbon fixation pathway are carboxylation reactions.

According to some embodiments of the invention, one of the reactions ofthe carbon fixation pathway is a transcarboxylation reaction.

According to some embodiments of the invention, the enzyme whichperforms the transcarboxylation reaction is methylmalonyl-CoAcarboxytransferase.

According to some embodiments of the invention, the products of thereactions of the carbon fixation pathway further comprise pyruvate,phophoenolpyruvate (PEP), malate, malyl CoA and acetyl CoA.

According to some embodiments of the invention, the system is expressedin cells.

According to some embodiments of the invention, the system is expressedin eukaryotic cells.

According to some embodiments of the invention, the system is expressedin prokaryotic cells.

According to some embodiments of the invention, the system is present ina reactor.

According to some embodiments of the invention, the cells are selectedfrom the group consisting of bacteria cells, algae cells and higherplant cells.

According to some embodiments of the invention, the bacteria cells areE. coli cells.

According to some embodiments of the invention, the bacteria cellsexpress Pyruvate Dikinase, PEP Carboxylase, Malate Dehyderogenase,Lactate Dehydrogenase, Glyoxylate Carboligase, Tartronate-SemialdehydeReductase, Glycerate Kinase, Malyl-CoA Synthetase, Malyl-CoA Lyase,Methlmalonyl-CoA Carboxytransferase, Malonate SemialdehydeDehydrogenase, Alanine Aminomutase and Beta-Alanine PyruvateTransaminase.

According to some embodiments of the invention, the bacteria cellsexpress Pyruvate Dikinase, PEP Carboxylase, Malate Dehyderogenase,Lactate Dehydrogenase, Glyoxylate Carboligase, Tartronate-SemialdehydeReductase, Glycerate Kinase, Malyl-CoA Synthetase, Malyl-CoA Lyase,Methlmalonyl-CoA Carboxytransferase, Malonyl-CoA Reductase, PropionateCoA Transferase, Enoyl-CoA Hydratase and Lactoyl-CoA dehydratase.

According to some embodiments of the invention, the bacteria cellsfurther express NAD⁺-dependent formate dehydrogenase or NAD:phosphiteoxidoreductase.

According to some embodiments of the invention, the Malate Dehydrogenaseis a higher plant Malate dehydrogenase.

According to some embodiments of the invention, the lactateDehydrogenase is a trichomona lactate dehydrogenase.

According to some embodiments of the invention, the bacteria cells arecyanobacteria cells.

According to some embodiments of the invention, the cyanobacterial cellsexpress Pyruvate Dikinase, PEP Carboxylase, Malate Dehyderogenase,lactate dehydrogenase, Glyoxylate Carboligase, Tartronate-SemialdehydeReductase and Glycerate Kinase, Malyl-CoA Synthetase, Malyl-CoA Lyase,Methlmalonyl-CoA Carboxytransferase, Malonate SemialdehydeDehydrogenase, Alanine Aminomutase and Beta-Alanine PyruvateTransaminase.

According to some embodiments of the invention, the cyanobacterial cellsexpress Pyruvate Dikinase, PEP Carboxylase, Malate Dehyderogenase,lactate dehydrogenase, Glyoxylate Carboligase, Tartronate-SemialdehydeReductase and Glycerate Kinase, Malyl-CoA Synthetase, Malyl-CoA Lyase,Methlmalonyl-CoA Carboxytransferase, Malonyl-CoA Reductase, PropionateCoA Transferase, Enoyl-CoA Hydratase and Lactoyl-CoA dehydratase.

According to some embodiments of the invention, the Malate Dehydrogenaseis a higher plant Malate dehydrogenase.

According to some embodiments of the invention, the lactateDehydrogenase is a trichomona lactate dehydrogenase.

According to some embodiments of the invention, the algae cells areChlamydomonas reinhardtii cells.

According to some embodiments of the invention, the Chlamydomonasreinhardtii cells express PEP Carboxylase, Malate Dehyderogenase,Glycerate Kinase, Pyruvate Dikinase, Malyl-CoA Synthetase, Malyl-CoALyase, Methlmalonyl-CoA Carboxytransferase, Malonate SemialdehydeDehydrogenase, Alanine Aminomutase, Beta-Alanine Pyruvate Transaminase,Glyoxylate Carboligase and Tartronate-Semialdehyde Reductase.

According to some embodiments of the invention, the Chlamydomonasreinhardtii cells express PEP Carboxylase, Malate Dehyderogenase,Glycerate Kinase, Pyruvate Dikinase, Malyl-CoA Synthetase, Malyl-CoALyase, Methlmalonyl-CoA Carboxytransferase, Malonyl-CoA Reductase,Propionate CoA Transferase, Enoyl-CoA Hydratase, Lactoyl-CoAdehydratase, Lactate Dehydrogenase, Glyoxylate Carboligase andTartronate-Semialdehyde Reductase.

According to some embodiments of the invention, the higher plant cell isa tobacco cell.

According to some embodiments of the invention, the tobacco cellsexpress Pyruvate Dikinase, PEP Carboxylase, Malate Dehydrogenase,Glycerate Kinase, Malyl-CoA Synthetase, Malyl-CoA Lyase,Methlmalonyl-CoA Carboxytransferase, Malonate SemialdehydeDehydrogenase, Alanine Aminomutase, Beta-Alanine Pyruvate Transaminase,Glyoxylate Carboligase and Tartronate-Semialdehyde Reductase.

According to some embodiments of the invention, the tobacco cellsexpress Pyruvate Dikinase, PEP Carboxylase, Malate Dehydrogenase,Glycerate Kinase, Malyl-CoA Synthetase, Malyl-CoA Lyase,Methlmalonyl-CoA Carboxytransferase, Malonyl-CoA Reductase, PropionateCoA Transferase, Enoyl-CoA Hydratase, Lactoyl-CoA dehydratase, LactateDehydrogenase, Glyoxylate Carboligase and Tartronate-SemialdehydeReductase.

According to some embodiments of the invention, the system furthercomprises an electron donor.

According to some embodiments of the invention, the electron donor isselected from the group consisting of ATP, NADH and NADPH.

According to some embodiments of the invention, the system is in aparticle selected from the group consisting of polymeric particles,microcapsules liposomes, microspheres, microemulsions, nano-plates,nanoparticles, nanocapsules and nano spheres.

According to some embodiments of the invention, the enzymes areencapsulated within the particle.

According to some embodiments of the invention, the enzymes are embeddedwithin the particle.

According to some embodiments of the invention, the enzymes are adsorbedon a surface of the particle.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a system of one embodiment ofthe present invention, the method comprising expressing in the bacteriaMalyl-CoA Synthetase, Malyl-CoA Lyase, Methlmalonyl-CoACarboxytransferase, Malonate Semialdehyde Dehydrogenase, AlanineAminomutase and Beta-Alanine Pyruvate Transaminase, thereby generatingthe system of one embodiment of the present invention.

According to some embodiments of the invention, the Malyl-CoASynthetase, Malyl-CoA Lyase, Methlmalonyl-CoA Carboxytransferase,Malonate Semialdehyde Dehydrogenase, Alanine Aminomutase andBeta-Alanine Pyruvate Transaminase are prokaryotic.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a system of one embodiment ofthe present invention, the method comprising expressing in the bacteriaMalyl-CoA Synthetase, Malyl-CoA Lyase, Methlmalonyl-CoACarboxytransferase, Malonyl-CoA Reductase, Propionate CoA Transferase,Enoyl-CoA Hydratase and Lactoyl-CoA dehydratase, thereby generating thesystem of one embodiment of the present invention.

According to some embodiments of the invention, the method furthercomprises expressing in the bacteria a higher plant malate dehydrogenaseand a Trichomona lactate dehydrogenase.

According to some embodiments of the invention, the method furthercomprises expressing in the bacteria NAD⁺-dependent formatedehydrogenase or NAD:phosphite oxidoreductase.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a system of one embodiment ofthe present invention, the method comprising expressing in the bacteriaMalyl-CoA Synthetase, Malyl-CoA Lyase, Methlmalonyl-CoACarboxytransferase, Malonate Semialdehyde Dehydrogenase, AlanineAminomutase and Beta-Alanine Pyruvate Transaminase, thereby generatingthe system of one embodiment of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating the system of one embodiment ofthe present invention, the method comprising expressing in the bacteriaMalyl-CoA Synthetase, Malyl-CoA Lyase, Methlmalonyl-CoACarboxytransferase, Malonyl-CoA Reductase, Propionate CoA Transferase,Enoyl-CoA Hydratase and Lactoyl-CoA dehydratase, thereby generating thesystem of thereby generating the system of one embodiment of the presentinvention.

According to some embodiments of the invention, the method furthercomprises expressing in the bacteria Lactate Dehydrogenase.

According to some embodiments of the invention, the lactatedehydrogenase is a Trichomona lactate dehydrogenase.

According to some embodiments of the invention, the method furthercomprises expressing in the bacteria a higher plant MalateDehydrogenase.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating the system of one embodiment ofthe present invention comprising expressing enzymes in the cell, theenzymes being PEP Carboxylase, Malate Dehydrogenase, Glycerate Kinase,Pyruvate Dikinase, Malyl-CoA Synthetase, Malyl-CoA Lyase,Methlmalonyl-CoA Carboxytransferase, Malonate SemialdehydeDehydrogenase, Alanine Aminomutase, Beta-Alanine Pyruvate Transaminase,Glyoxylate Carboligase and Tartronate-Semialdehyde Reductase, whereinthe enzymes are targeted to the chloroplast, thereby generating thesystem of thereby generating the system of one embodiment of the presentinvention.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating the system of one embodiment ofthe present invention comprising expressing enzymes in the cell, theenzymes being PEP Carboxylase, Malate Dehydrogenase, Glycerate Kinase,Pyruvate Dikinase, Malyl-CoA Synthetase, Malyl-CoA Lyase,Methlmalonyl-CoA Carboxytransferase, Malonyl-CoA Reductase, PropionateCoA Transferase, Enoyl-CoA Hydratase, Lactoyl-CoA dehydratase, LactateDehydrogenase, Glyoxylate Carboligase and Tartronate-SemialdehydeReductase, wherein the enzymes are targeted to the chloroplast, therebygenerating the system of thereby generating the system of one embodimentof the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating the system of one embodiment ofthe present invention, the method comprising expressing in the cellsenzymes, the enzymes being Pyruvate Dikinase, PEP Carboxylase, MalateDehyderogenase, Glycerate Kinase, Malyl-CoA Synthetase, Malyl-CoA Lyase,Methlmalonyl-CoA Carboxytransferase, Malonate SemialdehydeDehydrogenase, Alanine Aminomutase, Beta-Alanine Pyruvate Transaminase,Glyoxylate Carboligase and Tartronate-Semialdehyde Reductase, whereinthe enzymes are targeted to the chloroplast, thereby generating thesystem of one embodiment of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating the system of one embodiment ofthe present invention, the method comprising expressing in the cellsenzymes, the enzymes being Pyruvate Dikinase, PEP Carboxylase, MalateDehyderogenase and Glycerate Kinase, Malyl-CoA Synthetase, Malyl-CoALyase, Methlmalonyl-CoA Carboxytransferase, Malonyl-CoA Reductase,Propionate CoA Transferase, Enoyl-CoA Hydratase, Lactoyl-CoAdehydratase, Lactate Dehydrogenase, Glyoxylate Carboligase andTartronate-Semialdehyde Reductase wherein the enzymes are targeted tothe chloroplast, thereby generating the system of one embodiment of thepresent invention.

According to an aspect of some embodiments of the present inventionthere is provided a system for carbon fixation, as exemplified in FIGS.7A-G and 9A-N.

According to an aspect of some embodiments of the present inventionthere is provided an autotrophic E. coli expressing enzymes of theCalvin-Benson cycle.

According to some embodiments of the invention, the autotrophic E. coliexpress phosphoribulokinase and Ribulose-Bisphosphate Carboxylase.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a diagram illustrating natural carbon fixation pathways. Shownis the variety and interrelationships of four of the five alternativesto the Calvin Benson cycle. A fifth option, the reductive acetyl-CoApathway is not shown. A color notation has been used to display thedifferent functional groups composing the metabolites, which alsocorresponds to the oxidation states of the carbons: red circles indicatecarboxyl, purple corresponds to carbonyl, green to hydroxyl, azure toamine and black to hydrocarbon. The ‘═’ mark symbolizes a double bond.UQ and MQ correspond to ubiquinone and menaquinone, respectively.

Enzymes: (A1) 2-Ketoglutarate Synthase; (A2) Isocitrate Dehydrogenase;(A3) Aconitate Hydratase; (A4) ATP Citrate Lyase; (B1) PyruvateSynthase; (B2) Pyruvate Water (Phosphate) Dikinase; (B3) PEPCarboxylase; (B4) Malate Dehydrogenase; (B5) Fumarate Hydratase; (B6)Fumarate Reductase; (B7) Succinyl-CoA Synthetase; (C1) Succinyl-CoAReductase; (C2) 4-Hydroxybutyrate Dehydrogenase; (C3)4-Hydroxybutyryl-CoA Synthetase; (C4) 4-Hydroxybutyryl-CoA Dehydratase;(C5) Enoyl-CoA Hydratase (Crotonase); (C6) 3-Hydroxybutyryl-CoADehydrogenase; (C7) Acetyl-CoA C-Acyltransferase; (D1) Acetyl-CoACarboxylase (D2) Malonyl-CoA Reductase; (D3) Propionyl-CoA synthase;(D4) propionyl-CoA Carboxylase; (D5) Methylmalonyl-CoA Epimerase; (D6)Methylmalonyl-CoA Mutase; (E1) Succinyl-CoA Synthetase; (E2) SuccinateDehydrogenase; (E3) Fumarate Hydratase; (E4) Malyl-CoA Synthetase and(E5) Malyl-CoA Lyase. E.C. numbers are given in Example 2.

FIGS. 2A-2C are diagrams illustrating thermodynamic constraints oncarbon fixation. The minimal number of ATP molecules that should behydrolyzed to ensure that carbon fixation would be thermodynamicallyfeasible. The cycle's product was assumed to be GA3P. Different electrondonors alternatives are analyzed at varying conditions of pH, ionicstrength and under ambient CO₂ ^(gas) concentration (390 ppm). (A) all 6of the electron donors are NAD(P)H, as in the case of the reductivepentose phosphate pathway. (B) two of the electron donors areferredoxins, one is menaquinone (MQ) and the other three are NAD(P)H, asin the case of the rTCA cycle, where MQ is the electron donor forfumarate reductase (other electron donors are shown in FIG. 1B). (C) Twoof the electron donors are ferredoxins (Fd) and the other four areNAD(P)H, as in the case of the rAcCoA pathway. Bold lines (right andmiddle schemes) correspond to the feasibility ranges of the pathways, asdictated by their actual ATP consumption (5 ATP molecules by the rTCAcycle and 4 by the rAcCoA pathway). The pathways are not feasible at pHand ionic strength values corresponding to the areas to the right ofthose lines; the ATP requirement in those areas is higher than thatconsumed by the pathways. The rPP cycle consumes 9 ATP molecules, wellabove the minimal thermodynamic requirement. See Example 2 for furtherdetails and calculations.

FIG. 3 is a diagram of the shortest possible carbon fixation cycle—a‘metabolic shortcut’ of the rTCA cycle. Two CO₂ molecules are fixed togive a two carbon oxalate product as the cycle product. Every circledenotes carbon atom. A color notation has been used to display thedifferent functional groups composing the metabolites, which alsocorresponds to the oxidation states of the carbons: red circles indicatecarboxyl, purple corresponds to carbonyl, green to hydroxyl, azure toamine and black to hydrocarbon. The ‘═’ mark symbolizes a double bond.UQ and MQ correspond to ubiquinone and menaquinone, respectively. Asexplained in the text, this cycle is not thermodynamically feasible anddoes not represent a viable alternative for carbon fixation.

Enzymes: (1) Succinyl-CoA Synthetase; (2) 2-Ketoglutarate Synthase; (3)Isocitrate Dehydrogenase and (4) Isocitrate Lyase. E.C. numbers aregiven in Example 2

FIGS. 4A-4C are diagrams of carbon fixation pathways according toembodiments of the present invention. (A,B) The C4-Glyoxylate cycles.Promising carbon fixation pathways utilizing the most favorablecarboxylating enzyme, PEP carboxylase in a nested manner to achieve acycle resulting in a glyoxylate product easily transformed into GASP.Coloring and symbols are as in FIG. 3. E.C. numbers given in Example 2.(C) The metabolic overlap between the natural C4 module (marked inblack) and the synthetic C4-glyoxylate cycles (additional reactionsmarked in red).

(A) Enzymes: (1) Pyruvate Water (Phosphate) Dikinase; (2) PEPCarboxylase; (3) Malate Dehydrogenase; (4) Malyl-CoA Synthetase; (5)Malyl-CoA Lyase; (6) Methylmalonyl-CoA Carboxytransferase; (7)Malonyl-CoA Reductase (malonate-semialdehyde forming); (8)beta-Alanine-Pyruvate Transaminase and (9) Alanine 2,3-Aminomutase.

(B) Enzymes: (1-6) as in (A); (7) Malonyl-CoA Reductase(3-hydroxypropionate forming); (8) Propionate CoA Transferase; (9)Enoyl-CoA Hydratase; (10) Lactoyl-CoA Dehydratase and (11) LactateDehydrogenase.

FIG. 5A is a scheme of the reductive pentose phosphate cycle, asinspired from Poolman M G et al 2000, J Exp Bot 51 Spec No: 319-328.

Dashed line corresponds to RUBISCO's oxygenase reaction and to thephotorespiration pathway. See Examples section.Glyceraldehyde-3-Phosphate is considered as the pathway product.

Enzymes: (1) RUBISCO; (2) Phosphoglycerate Kinase; (3) Glyceraldehyde-3PDehydrogenase (Phosphorylating); (4) Triose-Phosphate Isomerase; (5)Fructose-Bisphosphate Aldolase; (6) Fructose-Bisphosphatase; (7)Transketolase; (8) Aldolase (Fructose-Bisphosphate Aldolase); (9)Sedoheptulose-Bisphosphatase; (10) Transketolase; (11)Ribose-5-Phosphate Isomerase; (12) Ribulose-Phosphate 3-Epimerase; (13)Phosphoribulokinase. E.C. numbers are given in Example 2.

FIG. 5B is a scheme of the reductive acetyl-CoA pathway, as inspiredfrom (Drake H L, Kirsten K, & Matthies C (2006) Acetogenic ProkaryotesThe Prokaryotes, (Springer New York), pp 354-420). THF corresponds totetrahydrofolate.

Enzymes: (1) Formate Dehydrogenase; (2) FormyltetrahydrofolateSynthetase; (3) Methenyltetrahydrofolate Cyclohydrolase; (4)Methenyltetrahydrofolate Dehydrogenase; (5) MethenyltetrahydrofolateReductase; (6) CO dehydrogenase/acetyl-CoA synthase.

FIG. 6 is a scheme of assimilation pathways. Those pathways convert theproducts of the different cycles (highlighted in pink) to the commonproduct glyceraldehyde-3-phosphate.

Enzymes: (1) Pyruvate Synthase; (2) Pyruvate Water (Phosphate)Dikinases; (3) Enolase; (4) Phosphoglycerate Mutase; (5)Phosphoglycerate Kinase; (6) Glyceraldehyde-3P Dehydrogenase(Phosphorylating); (7) Methylmalyl-CoA Lyase; (8) Mesaconyl-CoAHydratase; (9) Un-known; (10) Succinate:Citramalate CoA-Transferase;(11) Citramalyl-CoA Lyase; (12) Glyoxylate Carboligase; (13)Tartronate-Semialdehyde Reductase and (14) Glycerate Kinase.

The coloring scheme is identical to that of FIG. 1. E.C. numbers aregiven in Example 2.

FIGS. 7A-7G are diagrams of the simplest carbon fixation cycles.

The KGS-ICDH and PyrS-ME pathways are not thermodynamically feasiblebecause they contain a thermodynamic distributed bottleneck (see text).The KGS-KGC and PyrS-PyrC-Glyoxylate pathways are thermodynamicallyquestionable; the free energy change associated with their cycles may bepositive under a broad range of estimated physiological concentrationsof their product (see text).

(7A) The KGS-ICDH cycle.

Enzymes: (1) Succinyl-CoA Synthetase; (2) 2-Ketoglutarate Synthase; (3)Isocitrate Dehydrogenase and (4) Isocitrate Lyase.

(7B) The KGS-KGC cycle.

Enzymes: (1) Succinyl-CoA Synthetase; (2) 2-Ketoglutarate Synthase; (3)2-ketoglutarate carboxylase; (4) isocitrate:NADP oxidoreductase and (5)Isocitrate Lyase.

(7C) The PyrS-ME cycle.

Enzymes: (1) Pyruvate Synthase; (2) ‘Malic’ enzyme; (3) Malyl-CoASynthetase and (4) Malyl-CoA Lyase.

(7D) The PyrS-PyrC-Glyoxylate cycle.

Enzymes: (1) Pyruvate Synthase; (2) Pyruvate Carboxylase; (3) MalateDehydrogenase; (4) Malyl-CoA Synthetase and (5) Malyl-CoA Lyase.

(7E) The PyrS-PEPC-Glyoxylate cycle.

Enzymes: (1) Pyruvate Synthase; (2) Pyruvate Water (Phosphate) Dikinase;(3) PEP Carboxylate; (4) Malate Dehydrogenase; (5) Malyl-CoA Synthetaseand (6) Malyl-CoA Lyase.

(7F) The PyrS-PyrC-Oxalate cycle.

Enzymes: (1) Acetyl-CoA Synthetase; (2) Pyruvate Synthase; (3) PyruvateCarboxylase; (4) Oxaloacetase; (5) Oxalyl-CoA Synthetase and (6)Glyoxylate Dehydrogenase (acylating).

(7G) The PyrS-PEPC-Oxalate cycle.

Enzymes: (1) Acetyl-CoA Synthetase; (2) Pyruvate Synthase; (3) PyruvateWater (Phosphate) Dikinases; (4) PEP Carboxylase; (5) Oxaloacetase; (6)Oxalyl-CoA Synthetase and (7) Glyoxylate Dehydrogenase (acylating).

The coloring scheme is identical to that of FIG. 1. E.C. numbers aregiven in Example 2.

FIG. 8 is a diagram of the core of the most promising carbon fixationpathways family and its modules. The metabolic blueprint of the MOGpathways alongside different metabolic alternatives they can employ. Theenzymes lactoyl-CoA dehydratase and alanine 2,3-aminomutase (see below)present several difficulties as explained in detailed in Example 2.

Enzymes of the core structure: (1) Malate Dehydrogenase; (2) Malyl-CoASynthetase and (3) Malyl-CoA Lyase.

Module A: (C1) Pyruvate Water (Phosphate) Dikinase; (C2) PEP Carboxylaseand (C3) Pyruvate Carboxylase.

Module B: (A1) Acetyl-CoA Carboxylase and (A2) Methylmalonyl-CoACarboxytransferase.

Module C: (B1) Malonyl-CoA Reductase (3-hydroxypropionate forming); (B2)Propionate CoA Transferase; (B3) Enoyl-CoA Hydratase; (B4) Lactoyl-CoADehydratase; (B5) Lactate Dehydrogenase; (B6) Malonyl-CoA Reductase(malonate-semialdehyde forming); (B7) beta-Alanine-Pyruvate Transaminaseand (B8) Alanine 2,3-Aminomutase.

Coloring and symbols are as in FIG. 1.

FIGS. 9A-9N are diagrams of other carbon fixation pathways.

(9A) The AcC-ICDH-Glycerate pathway.

Enzymes: (1) Acetyl-CoA Carboxylase; (2) Malonyl-CoA Reductase; (3)Propionyl-CoA synthase; (4) 2-Hydroxyglutarate Synthase; (5)2-Hydroxyglutarate Dehydrogenase; (6) Isocitrate Dehydrogenase; (7)Isocitrate Lyase; (8) Succinate Dehydrogenase; (9) Fumarate Hydratase;(10) Malyl-CoA Synthetase and (11) Malyl-CoA Lyase.

(9B) The AcC-ICDH-Citrate pathway.

Enzymes: (1) Acetyl-CoA Carboxylase; (2) Malonyl-CoA Reductase; (3)Propionyl-CoA synthase; (4) 2-Hydroxyglutarate Synthase; (5)2-Hydroxyglutarate Dehydrogenase; (6) Isocitrate Dehydrogenase; (7)Aconitate Hydratase; (8) ATP Citrate Lyase; (9) Malate Dehydrogenase;(10) Malyl-CoA Synthetase and (11) Malyl-CoA Lyase.

(9C) The AcC-ICDH-Citrate/Pyruvate pathway.

Enzymes: (1) Acetyl-CoA Carboxylase; (2) Malonyl-CoA Reductase; (3)Propionyl-CoA synthase; (4) 2-Hydroxyglutarate Synthase; (5)2-Hydroxyglutarate Dehydrogenase; (6) Isocitrate Dehydrogenase; (7)Aconitate Hydratase; (8) ATP Citrate Lyase; (9) Malate Dehydrogenase;(10) Malyl-CoA Synthetase; (11) Malyl-CoA Lyase; (12) AconitateHydratase; (13) ATP Citrate Lyase and (14) Methylmalonyl-CoACarboxytransferase.

(9D) The AcC-PrC-Glycerate pathway.

Enzymes: (1) Acetyl-CoA Carboxylase; (2) Malonyl-CoA Reductase; (3)Propionyl-CoA synthase; (4) Propionyl-CoA Carboxylase; (5)Methylmalonyl-CoA Epimerase; (6) Methylmalonyl-CoA Mutase; (7)Succinyl-CoA Synthetase; (8) Succinate Dehydrogenase; (9) Fumarase; (10)Malyl-CoA Synthetase and (11) Malyl-CoA Lyase.

(9E) The AcC-PrC-Citrate pathway.

Enzymes: (1) Acetyl-CoA Carboxylase; (2) Malonyl-CoA Reductase; (3)Propionyl-CoA synthase; (4) Propionyl-CoA Carboxylase; (5)Methylmalonyl-CoA Epimerase; (6) Methylmalonyl-CoA Mutase; (7)Succinyl-CoA Synthetase; (8) Isocitrate Lyase; (9) Aconitate Hydratase;(10) ATP Citrate Lyase; (11) Malate Dehydrogenase; (12) Malyl-CoASynthetase and (13) Malyl-CoA Lyase.

(9F) The AcC-PrC-Citrate/Pyruvate pathway.

Enzymes: (1) Acetyl-CoA Carboxylase; (2) Malonyl-CoA Reductase; (3)Propionyl-CoA synthase; (4) Propionyl-CoA Carboxylase; (5)Methylmalonyl-CoA Epimerase; (6) Methylmalonyl-CoA Mutase; (7)Succinyl-CoA Synthetase; (8) Succinate Dehydrogenase; (9) FumarateHydratase; (10) Malyl-CoA Synthetase; (11) Malyl-CoA Lyase; (12)Isocitrate Lyase; (13) Aconitate Hydratase; (14) ATP Citrate Lyase and(15) Methylmalonyl-CoA Carboxytransferase.

(9G) The AcC-PrC-4-Hydroxybutyrate (no ferredoxin) pathway.

Enzymes: (1) Acetyl-CoA Carboxylase; (2) Malonyl-CoA Reductase; (3)Propionyl-CoA synthase; (4) Propionyl-CoA Carboxylase; (5)Methylmalonyl-CoA Epimerase; (6) Methylmalonyl-CoA Mutase; (7)Succinyl-CoA Reductase; (8) 4-Hydroxybutyrate Dehydrogenase; (9)4-Hydroxybutyryl-CoA Synthetase; (10) 4-Hydroxybutyryl-CoA Dehydratase;(11) Enoyl-CoA Hydratase (Crotonase); (12) 3-Hydroxybutyryl-CoADehydrogenase; (13) Acetyl-CoA C-Acyltransferase; (14) Succinyl-CoASynthetase; (15) Succinate Dehydrogenase; (16) Fumarate Hydratase and(17) Malic enzyme.

(9H) The CCR-PEPC pathway.

Enzymes: (1) Acetyl-CoA C-Acyltransferase; (2) 3-Hydroxybutyryl-CoADehydrogenase; (3) Enoyl-CoA Hydratase (Crotonase); (4) Crotonyl-CoACarboxylase/Reductase; (5) Ethylmalonyl-CoA Epimerase; (6)Ethylmalonyl-CoA Mutase; (7) Methylsuccinyl-CoA Dehydrogenase; (8)Un-known; (9) Succinate:Citramalate CoA-Transferase; (10) Citramalyl-CoALyase; (11) Pyruvate Water (Phosphate) Dikinases; (12) PEP Carboxylase;(13) Malate Dehydrogenase; (14) Malyl-CoA Synthetase and (15) Malyl-CoALyase.

(9I) The MCC-ICDH-4-Hydroxybutyrate pathway.

Enzymes: (1) Succinyl-CoA Synthetase; (2) Succinyl-CoA Reductase; (3)4-Hydroxybutyrate Dehydrogenase; (4) 4-Hydroxybutyryl-CoA Synthetase;(5) 4-Hydroxybutyryl-CoA Dehydratase; (6) Methylcrotonyl-CoACarboxylase; (7) (R)-2-Hydroxyglutaryl-CoA Dehydratase; (8) GlutaconateCoA-Transferase; (9) 2-Hydroxyglutarate Dehydrogenase; (10) IsocitrateDehydrogenase and (11) Isocitrate Lyase.

(9J) The MCC-ICDH-Citrate pathway.

Enzymes: (1) Acetyl-CoA C-Acyltransferase; (2) 3-Hydroxybutyryl-CoADehydrogenase; (3) Enoyl-CoA Hydratase (Crotonase); (4)Methylcrotonyl-CoA Carboxylase; (5) (R)-2-Hydroxyglutaryl-CoADehydratase; (6) Glutaconate CoA-Transferase; (7) 2-HydroxyglutarateDehydrogenase; (8) Isocitrate Dehydrogenase; (9) Aconitate Hydratase;(10) ATP Citrate Lyase; (11) Malate Dehydrogenase; (12) Malyl-CoASynthetase and (13) Malyl-CoA Lyase.

(9K) The PyrS-PEPC-KGS-Glutamate pathway and the AcC-PrC-KGS-Glutamatepathway.

Enzymes: (1) Acetyl-CoA Carboxylase; (2) Malonyl-CoA Reductase; (3)Propionyl-CoA synthase; (4) Propionyl-CoA Carboxylase; (5)Methylmalonyl-CoA Epimerase; (6) Methylmalonyl-CoA Mutase; (7) PyruvateSynthase; (8) Pyruvate Water (Phosphate) Dikinase; (9) PEP Carboxylase;(10) Malate Dehydrogenase; (11) Fumarate Hydratase; (12) SuccinateDehydrogenase; (13) Succinyl-CoA Synthetase; (14) 2-KetoglutarateSynthase (15) Glutamate Dehydrogenase; (16) Glutamate Mutase; (17)Methylaspartate Ammonia-Lyase; (18) 2-Methylmalate Dehydratase; (19)Succinate:Citramalate CoA-Transferase and (20) Citramalyl-CoA Lyase.

(9L) The PrC-KGS-Glutamate pathway.

Enzymes: (1) Propionyl-CoA Carboxylase; (2) Methylmalonyl-CoA Epimerase;(3) Methylmalonyl-CoA Mutase; (4) 2-Ketoglutarate Synthase (5) GlutamateDehydrogenase; (6) Glutamate Mutase; (7) Methylaspartate Ammonia-Lyase;(8) Un-Known; (9) Mesaconyl-CoA Hydratase and (10) Methylmalyl-CoALyase.

(9M) The PyrS-KGS-Glutamate pathway.

Enzymes: (1) Pyruvate synthase; (2) Citramalate Synthase; (3) MesaconateHydratase; (4) Methylaspartate Ammonia-Lyase; (5) Glutamate Mutase; (6)Glutamate Dehydrogenase; (7) Isocitrate Dehydrogenase; (8) AconitateHydratase; (9) ATP Citrate Lyase; (10) Malate Dehydrogenase; (11)Malyl-CoA Synthetase and (12) Malyl-CoA Lyase.

(9N) The PyrS-PEPC-Threonine pathway and the AcC-PEPC-Threonine pathway.

Enzymes: (1) Acetyl-CoA Carboxylase; (2) Malonyl-CoA Reductase(malonate-semialdehyde forming); (3) beta-Alanine-alpha-AlanineTransaminase; (4) Alanine Aminomutase; (5) Pyruvate Synthase; (6)Pyruvate Water (Phosphate) Dikinase; (7) PEP Carboxylase; (8) AspartateTransaminase; (9) Aspartate Kinase; (10) Aspartate-SemialdehydeDehydrogenase; (11) Homoserine Dehydrogenase; (12) Homoserine Kinase;(13) Threonine Synthase; (14) Threonine Aldolase; (15) AcetaldehydeDehydrogenase (acetylating); (16) L-Threonine 3-Dehydrogenase; (17)Glycine C-Acetyltransferase and (18) Glycine Transaminase.

The coloring scheme is identical to that of FIG. 1. E.C. numbers aregiven in Example 2.

FIGS. 10A-10C are diagrams illustrating the thermodynamics of carbonfixation pathways.

-   (A) Total dissolved inorganic carbon (CO₂ ^(aq)+HCO₃ ⁻+CO₃ ²⁻+H₂CO₂    ^(aq)) as a function of pH and ionic strength, under ambient CO₂    ^(gas) concentration, 387 ppm.-   (B) Minimal ATP requirement and feasibility of carbon fixation    pathways. The common product was assumed to be GASP. Different    electron donors schemes are contrasted as varying conditions of pH,    ionic strength and CO₂ ^(gas) concentrations. First row: all 6 of    the electron donors are NAD(P)H, as in the case of the reductive    pentose phosphate pathway. Second row: 3 of the electron donors are    ferredoxins (Fd) and the other 3 are NAD(P)H, as in the case of the    rAcCoA pathway. Third row: 2 of the electron donors are ferredoxins,    1 is menaquinone (MQ) and the other 3 are NAD(P)H, as in the case of    the rTCA cycle, where menaquinone is the electron donor for fumarate    reductase. Forth row: 2 of the electron donors are ferredoxins, 1 is    FAD and the other 3 are NAD(P)H, as in the case of the rTCA cycle,    where FAD is the electron donor for fumarate reductase. Fifth row: 2    of the electron donors are ferredoxins and the other 4 are NAD(P)H,    as in the case of the rTCA cycle, where NADH is the direct electron    donor for fumarate reductase. Columns correspond to different CO₂    ^(gas) concentrations. Bold lines correspond to the feasibility    ranges of the pathways: the pathways are not feasible at pH and    ionic strength values corresponding to the area to the right of    those lines.-   (C) Feasibility of carbon fixation cycles, as separate metabolic    units within the pathways, as function of pH, ionic strength and    different concentrations of the cycles' product, glyoxylate. Blue    represents the feasibility range (ΔG<0), while red corresponds to    infeasibility of carbon fixation (ΔG>0). The upper boxes correspond    to the ferredoxin-oxidoreductase pathways KGS-KGC,    PyrS-PyrC-Glyoxylate and PrC-KGS-Glutamate. The bottom boxes    correspond to the non-ferredoxin-oxidoreductase-containing pathway    MCC-ICDH-Citrate.

FIG. 11 is a graph illustrating pathway specific activity as a functionof the number of enzymes.

FIG. 12 is a table (Table 1) comparing carbon fixation pathways.

† The pathway specific activities of the ferredoxine-oxidoreductasepathways are artificially high because their calculations do not includethe specific activities of the ferredoin-oxidoreductase enzymes; seemain text.

(a) Pathway notation and a comprehensive discussion is given in Example2.

(b) AcC: acetyl-CoA carboxylase; PEPC: PEP carboxylase; PrC:propionyl-CoA carboxylase; ICDH: isocitrate dehydrogenase; CCR:crotonyl-CoA carboxylase/reductase and MMC: methylcrotonyl-CoAcarboxylase.

(c) See Example 2 and FIGS. 10A-10C for further details on theenergetics of the carbon fixation pathways.

(d) The specific activity of the enzyme alanine 2,3-aminomutase is notknown, and therefore not included in the calculation of the pathwayspecific activity, which might be somewhat lower.

(e) Refers to a pathway using the enzymes PEP caboxylase and pyruvatedikinase. The values given in green, at the raw below, refer to apathway utilizing the enzyme pyruvate carboxylase.

(f) The enzyme which converts mesaconyl-CoA into citramalate is notknown. Therefore, its specific activity was not included in thecalculation of the specific activity of the pathway, which might besomewhat lower.

(g) The specific activity of the enzyme 2-hydroxyglutarate synthase isnot known, and therefore not included in the calculation of the pathwayspecific activity, which might be somewhat lower.

(h) The specific activity of the enzyme 4-hydroxybutyryl-CoA synthetaseis not known, and therefore not included in the calculation of thepathway specific activity, which might be somewhat lower.

(i) The enzyme which converts methylsuccinyl-CoA into mesaconyl-CoA isnot known. Therefore, its specific activity was not included in thecalculation of the specific activity of the pathway, which might besomewhat lower.

(j) The specific activity of the carboxylating enzyme 2-ketoglutaratecarboxylase is not known (see FIGS. 5A-5B) and therefore the pathwayspecific activity was not calculated.

(k) The specific activity of the enzyme oxalate CoA ligase is not known,and therefore not included in the calculation of the pathway specificactivity, which might be somewhat lower.

(l) The enzyme which converts mesaconate into mesaconyl-CoA is notknown. Therefore, its specific activity was not included in thecalculation of the specific activity of the pathway, which might besomewhat lower.

(m) The value out of the parentheses refers to a cycle in which thesuccinate dehydrogenase utilizes ubiquinone. The value inside of theparentheses refers to a cycle in which the succinate dehydrogenaseutilizes NADPH (a non-natural co-factor for this enzyme). See Example 2.

(n) The value out of the parentheses refers to a cycle in which theglyoxylate shunt utilizes ubiquinone or FAD. The value inside of theparentheses refers to a cycle in which the glyoxylate shunt utilizesNADPH. See Example 2.

FIG. 13 is a table (Table 2) comparing carboxylating enzymes.

(a) Several carboxylating enzymes were not evaluated: (1) “dead-end”carboxylating enzymes (carbamate kinase, urea carboxylase andcarbamoyl-phosphate synthase); (2) enzymes that are specific to highmolecular-weight-metabolites (indolepyruvate ferredoxin oxidoreductase,2-oxopropyl-CoM reductase, phosphoribosylaminoimidazole carboxylase andgeranoyl-CoA carboxylase) and (3) the reductive acetyl-CoA pathway'senzymes which does not fixate CO₂ and bicarbonate directly (carbonmonoxide dehydrogenase, formate dehydrogenase and acetyl-CoA synthase).

(b) Km and specific activity (saturating CO₂/HCO₃ ⁻) values werecollected from the literature. Specific activities in ambient CO₂/HCO₃ ⁻were calculated by assuming Michaelis-Menten kinetics with nocooperativity (See Example 2). For each enzyme and for all the threecriteria the worse half of values (the less optimized enzymes) wasdiscarded as well as the top 10% (which might present outliers anderrors in measurements) (see Methods). The table shows the average ofthe remaining values, and their range in parentheses.

(c) CO₂ concentration was estimated as 10 μM, while HCO₃ ⁻ concentrationwas conservatively estimated as 200 μM (Berg et al. Science 14 Dec.2007).

(d) Under common physiological conditions.

(e) Although the carbon species utilized in CO₂, affinity was measuredusing varied concentrations of bicarbonate.

(f) Values taken from one paper only.

(g) Scarcity of literature information about the carboxylation reaction,as well as extreme oxygen sensitivity disabled reliable specificactivity estimation.

(h) Values in Italic correspond to carboxylation rates of crotonyl-CoA(˜35% of methylcrotonyl-CoA).

(i) Rate of carboxylation, under saturating CO₂/HCO₃ ⁻, is higher thanthat of decarboxylation. However, under ambient CO₂/HCO₃ ⁻ thedecarboxylation rate is higher.

(j) The enzyme is inactivated by the direct action of O₂. However, theenzyme is operating (and remain active) in the mitochondria of Euglenagrown aerobically where it is stabilized by its co-factor: thiaminediphosphate.

(k) Although this enzyme is not a carboxylating one per se, it can beused instead of a true carboxylating enzyme (see FIG. 4).

FIG. 14 is a table providing enzyme kinetic details. Yellow shadingcorresponds to specific activity under substrate saturating conditions.Red shading represents the specific activity of enzymes under ambientinorganic carbon concentration, whereSA_(ambient)=SA_(saturating)*[C]/([C]+K_(m) ^(C)). [C] is the ambientconcentration of the carbon species and K_(m) ^(C) is the affinity ofthe enzyme towards that carbon species. Purple shading stand for thespecific activities of the ferredoxin-oxidoreductase enzymes, for whichkinetic data is scares in the carboxylation direction. Pink and blueshading correspond to ATP and NADPH costs, respectively.

FIG. 15 is a diagram illustrating the proposed (modified) Calvin-BensonCycle in E. coli (KEGG-style). Green boxes represent native enzymes,while red boxes correspond to foreign enzymes that should be expressedin the host.

FIG. 16 is a diagram of a carbon fixation pathway according toembodiments of the present invention. In this pathway, the enzymelactate-malate transhydrogenase (EC 1.1.99.7) is used, that couples theoxidation of lactate with the reduction of oxaloacetate.

Enzymes in the scheme: (1) Pyruvate Dikinase, (2) PEP Carboxylase, (3)Lactate-Malate Transhydrogenase, (4) Malyl-CoA Synthetase, (5) Malyl-CoALyase, (6) Methlmalonyl-CoA Carboxytransferase, (7) Malonyl-CoAReductase, (8) Propionate CoA Transferase, (9) Enoyl-CoA Hydratase, (10)Lactoyl-CoA dehydratase.

FIG. 17 is a diagram illustrating carbon fixation pathways according toembodiments of the present invention, in which inorganic carbon is fixedto pyruvate. The four common metabolites in all are acetyl-CoA,malonyl-CoA, pyruvate and oxaloacetate. The different modules andoptions are given in the attached scheme.

Enzymes in the scheme: (A1) Acetyl-CoA Carboxylase, (A2)Methlmalonyl-CoA Carboxytransferase, (B1) Malonyl-CoA Reductase, (B2)Propionate CoA Transferase, (B3) Enoyl-CoA Hydratase, (B4) Lactoyl-CoAdehydratase, (B5) Lactate Dehydrogenase, (B6) Malonate SemialdehydeDehydrogenase, (B7) Beta-Alanine Pyruvate Transaminase, (B8) AlanineAminomutase, (C1) Pyruvate Dikinase, (C2) PEP Carboxylase, (C3) PyruvateCarboxylase, (D1) Malate Dehydrogenase, (D2) Malyl-CoA Synthetase, (D3)Malyl-CoA Lyase, (D4) Glycine Dehydrogenase, (D5) 3-HydroxyaspartateAldolase, (D6) erythro-3-hydroxyaspartate ammonia-lyase, (D7) FumarateHydratase, (D8) Fumarate Reductase, (D9) Isocitrate Lyase, (D10)Aconitase, (D11) ATP-Citrate Lyase, (D12) Glycine Reductase, (D13)Phosphate Acetyltransferase, (D14) Succinate Thiokinase, (D15)Succinyl-CoA Reductase, (D16) Succinate Semialdehyde Reductase, (D17)4-Hydroxybutyryl-CoA Synthetase, (D18) 4-Hydroxybutyryl-CoA Dehydratase,(D19) Crotonyl-CoA Hydratase, (D20) 3-Hydroxybutyryl-CoA Dehydrogenaseand (D21) Acetoacetyl-CoA β-Ketothiolase.

FIG. 18 is a diagram of a PEPC-lactate/citramalate pathway according toembodiments of the present invention.

Enzymes for the PEPC-Lactate/Citramalate pathway: (1) Pyruvate Dikinase,(2) PEP Carboxylase, (3) Malate Dehydrogenase, (4) Malyl-CoA Synthetase,(5) Malyl-CoA Lyase, (6) Methlmalonyl-CoA Carboxytransferase, (7)Malonyl-CoA Reductase, (8) Propionate CoA Transferase, (9) Enoyl-CoAHydratase, (10) Lactoyl-CoA dehydratase, (11) Lalate Dehydrogenase, (12)Propionyl-CoA Synthethase, (13) L-malyl-CoA lyase, (14)B-methylamalyl-CoA lyase, (15) Mesaconyl-CoA C1-C4 Coa Transferase (16)Mesaconyl-C4-Coa Hydratase and (17) Citramalyl-CoA Lyase.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to enzymaticsystems for carbon fixation and methods of generating same.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Carbon fixation is the process by which carbon dioxide is incorporatedinto organic compounds. In the process of transforming sunlight intobiological fuel, plants absorb carbon dioxide using over 70% of thefresh water utilized by humanity and the majority of cultivatable landresources on earth. These figures point to the central place that carbonfixation by plants plays in our global ecological footprint.

Carbon fixation in plants and algae is achieved by the Calvin-BensonCycle. The productivity of the Calvin-Benson cycle is limited, undermany conditions, by the slow rate and lack of substrate specificity ofthe carboxylating enzyme Rubisco. Several lines of evidence indicatethat in-spite of its shortcomings, Rubisco might already be naturallyoptimized and hence its potential for improvement is very limited. Ascarbon fixation has been shown to limit growth rate in many studies, thepresent inventors sought to develop alternative pathways that cansupport carbon fixation with a higher rate in the efforts towardssustainability.

The present inventors systematically explored the space of possiblesynthetic carbon fixation pathways that can be assembled from all ˜4000known metabolic enzymes. The present inventors designed thiscomputational search using a constraint-based modeling framework thatanalyzed the possible metabolic pathways based on physicochemicalcriteria that include topology, energetics and kinetics.

Whilst reducing the present invention to practice, the present inventorsuncovered synthetic cycles with potential for superior characteristicsover natural ones. In particular, the present inventors found a newfamily of carbon fixation pathways that makes use of the most effectivecarboxylating enzyme, PEP carboxylase, and of the metabolic module usedin the efficient C4 plants (see FIGS. 4A-B and FIG. 8)

The present inventors propose implementing these pathways in varioussystems (cellular and non-cellular) and ultimately in photosyntheticorganisms including cyanobacteria (e.g. Synechococcus), algae (e.g.Chlamydomonas) and higher plants (e.g. Arabidopsis, Tobacco).

Thus, according to one aspect of the present invention there is provideda system for carbon fixation, comprising enzymes which catalyzereactions of a carbon fixation pathway, wherein at least one of thereactions of the carbon fixation pathway is a carboxylation reaction,wherein products of the reactions of the carbon fixation pathwaycomprise oxaloacetate and malonyl-CoA, wherein an enzyme which performsthe carboxylation reaction is selected from the group consisting ofphophoenolpyruvate (PEP) carboxlase, pyruvate carboxylase and acetyl-CoAcarboxylase and wherein an additional product of the carbon fixationpathway is glyoxylate.

The term “carbon fixation” as used herein refers to a process throughwhich gaseous carbon dioxide is converted into a solid compound.

As used herein the phrase “carbon fixation pathway” refers to a set ofmolecules (e.g. enzymes, electron donors, co-factors etc.) that togetherenable autotrophic carbon fixation. As such the system of this aspect ofthe present invention comprises enzymes which are positioned relative toone another such that they are able to function to cause carbonfixation.

The term “enzyme” as used herein refers to a “catalytically functionalbiomolecule,” which includes both whole native (or native-size)molecules and derivatives (e.g. genetic modifications) thereof.

Details of contemplated enzymes to be used according to this aspect ofthe present invention are provided in FIG. 14.

According to this aspect of the present invention at least one of thereactions in the pathway is a carboxylating reaction.

The term “carboxylation reaction” refers to a reaction in which in whichan in-organic carbon is introduced into a substrate to become acarboxylic acid group.

Enzymes capable of performing carboxylating reactions are provided inFIG. 13. According to this aspect of the present invention at least oneof the enzymes of the carbon fixation pathway is phophoenolpyruvate(PEP) carboxlase, pyruvate carboxylase or acetyl CoA carboxylase.

According to a particular embodiment of this aspect of the presentinvention the carboxylating enzyme is PEP carboxylase.

According to one embodiment, two of the reactions of the pathway arecarboxylating reactions.

According to still another embodiment, one of the reactions of thepathway is a transcarboxylating reaction.

The term “transcarboxylation reaction” refers to a reaction in which acarboxylic acid group is transferred from one metabolite to another one.

An Exemplary enzyme contemplated for the transcarboxylating reaction ismethylmalonlyl-CoA carboxytransferase.

As used herein, the phrase “export product” refers to a product of one(or more) of the reactions of the carbon fixation pathway which does notserve as a substrate for the other enzymes of the pathway.

According to one embodiment, the export product is glyoxylate.

According to another embodiment, the export product is pyruvate.

According to this aspect of the present invention, the pathway specificactivity is greater than 0.3 μmol-GA3P/min/mg. The pathway specificactivity (analogous to an enzyme's specific activity) is defined to bethe maximal rate of glyceraldehyde-3-phosphate (GA3P) formation by 1 mgof pathway total protein—as detailed in Example 2 herein below.

Exemplary carbon fixation pathways of the present invention which haveglyoxylate as the export product are illustrated in FIGS. 4A-B, FIG. 8and FIG. 16.

Exemplary carbon fixation pathways of the present invention which havepyruvate as the export product are illustrated in FIGS. 17 and 18.

According to one embodiment, the enzymes of the carbon fixation pathwaysof the present invention are expressed in cells. The cells may beeukaryotic (e.g. plant cells) or prokaryotic (e.g. bacterial cells).Such cells include cells of photosynthetic organisms (cyanobacteria,algae and higher plants), chemosynthetic organisms, and non-autotrophicorganisms (e.g. E. coli). According to this embodiment the enzymes whichtake part in the carbon fixation pathways are present in the samecomponent of the cell such that they are able to cooperate together tofulfill their role in the carbon fixation pathways.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,roots (including tubers), and plant cells, tissues and organs. The plantmay be in any form including suspension cultures, embryos, meristematicregions, callus tissue, leaves, gametophytes, sporophytes, pollen, andmicrospores. Plants that are particularly useful in the methods of theinvention include all plants which belong to the superfamilyViridiplantee, in particular monocotyledonous and dicotyledonous plantsincluding a fodder or forage legume, ornamental plant, food crop, tree,or shrub selected from the list comprising Acacia spp., Acer spp.,Actinidia spp., Aesculus spp., Agathis australis, Albizia amara,Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassicaspp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadabafarinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicumspp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomumcassia, Coffea arabica, Colophospermum mopane, Coronillia varia,Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp.,Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogonspp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davalliadivaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogonamplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloapyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp.,Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa,Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp,Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffheliadissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago saliva, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryzaspp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petuniaspp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photiniaspp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara,Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopiscineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis,Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhusnatalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitysvefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghumbicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusselssprouts, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugar beet, sugar cane, sunflower, tomato, squash tea, trees.Alternatively algae and other non-Viridiplantae can be used for themethods of the present invention.

It will be appreciated that the number of additional enzymes which haveto be exogenously expressed in a particular cell will depend on theenzymes which are naturally expressed in that cell type and on thesub-cellular location thereof.

In addition, depending on the system selected for carbon fixation, otherfactors must be generated or expressed in the system to ensure asufficient energy supply. Thus, for example in a non-cellular system ATPand NADH and/or NADPH should be provided as detailed further below. InE. coli, NAD is the preferred intermediate electron acceptor because itcan directly serve both as an electron donor for carbon fixation and asan energy producer when oxidized by E. coli's respiratory electronchain. The two best candidates for providing E. coli with reducing power(and energy) are formate and phosphite. The soluble enzymeNAD⁺-dependent formate dehydrogenase irreversibly oxidizes formate(E′⁰=−430 mV) and reduces NAD⁺-formate cannot be directly assimilated byE. coli. NAD:phosphite oxidoreductase irreversibly oxidizes phosphite tophosphate (E′⁰=−650 mV) and reduces NAD⁺.

Example 3 lists the specific enzymes and factors required to beexpressed/combined in 4 exemplary organisms—E. coli; Synechocystis sp.strain PCC6803 (cyanobacteria); Chlamydomonas reinhardtii (algae);Tobacco (Nicotiana) plant and in a non-cellular system.

To express the enzymes of the present invention using recombinanttechnology, a polynucleotide encoding the enzymes is ligated into anucleic acid expression vector, which comprises the polynucleotidesequence under the transcriptional control of a cis-regulatory sequence(e.g., promoter sequence) suitable for directing constitutive, tissuespecific or inducible transcription of the polypeptides of the presentinvention in the host cells.

Thus, the present invention contemplates isolated polynucleotidesencoding the enzymes of the present invention.

The phrase “an isolated polynucleotide” refers to a single or doublestranded nucleic acid sequence which is isolated and provided in theform of an RNA sequence, a complementary polynucleotide sequence (cDNA),a genomic polynucleotide sequence and/or a composite polynucleotidesequences (e.g., a combination of the above).

As used herein the phrase “complementary polynucleotide sequence” refersto a sequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.Such a sequence can be subsequently amplified in vivo or in vitro usinga DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and at leastpartially genomic. A composite sequence can include some exon sequencesrequired to encode the polypeptide of the present invention, as well assome intronic sequences interposing therebetween. The intronic sequencescan be of any source, including of other genes, and typically willinclude conserved splicing signal sequences. Such intronic sequences mayfurther include cis acting expression regulatory elements.

As mentioned hereinabove, polynucleotide sequences of the presentinvention are inserted into expression vectors (i.e., a nucleic acidconstruct) to enable expression of the recombinant polypeptide. Theexpression vector of the present invention includes additional sequenceswhich render this vector suitable for replication and integration inprokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).Typical cloning vectors contain transcription and translation initiationsequences (e.g., promoters, enhances) and transcription and translationterminators (e.g., polyadenylation signals).

According to one embodiment of this aspect of the present invention, thepolynucleotides of the present invention are expressed in cells of aphotosynthetic organism (e.g. higher plant, algae or cyanobacteria).

Examples of constitutive plant promoters include, but are not limited toCaMV35S and CaMV19S promoters, tobacco mosaic virus (TMV), FMV34Spromoter, sugarcane bacilliform badnavirus promoter, CsVMV promoter,Arabidpsis ACT2/ACT8 actin promoter, Arabidpsis ubiquitin UBQ 1promoter, barley leaf thionin BTH6 promoter, and rice actin promoter.

An inducible promoter is a promoter induced by a specific stimulus suchas stress conditions comprising, for example, light, temperature,chemicals, drought, high salinity, osmotic shock, oxidant conditions orin case of pathogenicity. Examples of inducible promoters include, butare not limited to, the light-inducible promoter derived from the pearbcS gene, the promoter from the alfalfa rbcS gene, the promoters DRE,MYC and MYB active in drought; the promoters INT, INPS, prxEa, Hahsp17.7G4 and RD21 active in high salinity and osmotic stress, and thepromoters hsr2O3J and str246C active in pathogenic stress.

These constructs can be introduced into plant cells using Ti plasmid, Riplasmid, plant viral vectors, direct DNA transformation, microinjection,electroporation, Biolistics (gene gun) and other techniques well knownto the skilled artisan. See, for example, Weissbach & Weissbach [Methodsfor Plant Molecular Biology, Academic Press, NY, Section VIII, pp421-463 (1988)]. Other expression systems such as insects and mammalianhost cell systems, which are well known in the art, can also be used bythe present invention.

It will be appreciated that other than containing the necessary elementsfor the transcription and translation of the inserted coding sequence(encoding the polypeptide), the expression construct of the presentinvention can also include sequences engineered to optimize stability,production, purification, yield or activity of the expressedpolypeptide.

According to one embodiment, the enzymes of the present invention areexpressed with chloroplast targeting peptides.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol.30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al.(1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhaoet al. (1995) J. Biol. Chem. 270(11):6081-6087); plastocyanin (Lawrenceet al. (1997) J. Biol. Chem. 272(33):20357-20363); chorismate synthase(Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.(1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol.Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481.

Various methods can be used to introduce the expression vector of thepresent invention into the host cell system. Such methods are generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press,Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, AnnArbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Plant cells may be transformed stabley or transiently with the nucleicacid constructs of the present invention. In stable transformation, thenucleic acid molecule of the present invention is integrated into theplant genome and as such it represents a stable and inherited trait. Intransient transformation, the nucleic acid molecule is expressed by thecell transformed but it is not integrated into the genome and as such itrepresents a transient trait.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNAinto plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; glass fibers or silicon carbide whiskertransformation of cell cultures, embryos or callus tissue, U.S. Pat. No.5,464,765 or by the direct incubation of DNA with germinating pollen,DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman,G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. Horsch et al. in Plant Molecular Biology Manual A5,Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementaryapproach employs the Agrobacterium delivery system in combination withvacuum infiltration. The Agrobacterium system is especially viable inthe creation of transgenic dicotyledenous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The mostcommon method of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transformedplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant. Therefore, itis preferred that the transformed plant be regenerated bymicropropagation which provides a rapid, consistent reproduction of thetransformed plants.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant. Micropropagationallows mass production of quality plant material in a short period oftime and offers a rapid multiplication of selected cultivars in thepreservation of the characteristics of the original transgenic ortransformed plant. The advantages of cloning plants are the speed ofplant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration ofculture medium or growth conditions between stages. Thus, themicropropagation process involves four basic stages: Stage one, initialtissue culturing; stage two, tissue culture multiplication; stage three,differentiation and plant formation; and stage four, greenhouseculturing and hardening. During stage one, initial tissue culturing, thetissue culture is established and certified contaminant-free. Duringstage two, the initial tissue culture is multiplied until a sufficientnumber of tissue samples are produced to meet production goals. Duringstage three, the tissue samples grown in stage two are divided and growninto individual plantlets. At stage four, the transformed plantlets aretransferred to a greenhouse for hardening where the plants' tolerance tolight is gradually increased so that it can be grown in the naturalenvironment.

Although stable transformation is presently preferred, transienttransformation of leaf cells, meristematic cells or the whole plant isalso envisaged by the present invention.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous nucleic acid sequences in plants is demonstrated bythe above references as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters (1990)269:73-76.

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous nucleic acid sequences such as thoseincluded in the construct of the present invention is demonstrated bythe above references as well as in U.S. Pat. No. 5,316,931.

In one embodiment, a plant viral nucleic acid is provided in which thenative coat protein coding sequence has been deleted from a viralnucleic acid, a non-native plant viral coat protein coding sequence anda non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral nucleic acid, andensuring a systemic infection of the host by the recombinant plant viralnucleic acid, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native nucleic acid sequencewithin it, such that a protein is produced. The recombinant plant viralnucleic acid may contain one or more additional non-native subgenomicpromoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or nucleic acid sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) nucleic acid sequencesmay be inserted adjacent the native plant viral subgenomic promoter orthe native and a non-native plant viral subgenomic promoters if morethan one nucleic acid sequence is included. The non-native nucleic acidsequences are transcribed or expressed in the host plant under controlof the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral nucleic acid isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that the sequences are transcribed or expressed in the host plantunder control of the subgenomic promoters to produce the desiredproduct.

In a fourth embodiment, a recombinant plant viral nucleic acid isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral nucleic acid to produce a recombinant plantvirus. The recombinant plant viral nucleic acid or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral nucleic acid is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(isolated nucleic acid) in the host to produce the desired protein.

In addition to the above, the nucleic acid molecule of the presentinvention can also be introduced into a chloroplast genome therebyenabling chloroplast expression.

A technique for introducing exogenous nucleic acid sequences to thegenome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theexogenous nucleic acid is introduced via particle bombardment into thecells with the aim of introducing at least one exogenous nucleic acidmolecule into the chloroplasts. The exogenous nucleic acid is selectedsuch that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the exogenous nucleic acid includes, inaddition to a gene of interest, at least one nucleic acid stretch whichis derived from the chloroplast's genome. In addition, the exogenousnucleic acid includes a selectable marker, which serves by sequentialselection procedures to ascertain that all or substantially all of thecopies of the chloroplast genomes following such selection will includethe exogenous nucleic acid. Further details relating to this techniqueare found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which areincorporated herein by reference. A polypeptide can thus be produced bythe protein expression system of the chloroplast and become integratedinto the chloroplast's inner membrane.

It will be appreciated that any of the construct types used in thepresent invention can be co-transformed into the same organism (e.g.plant) using same or different selection markers in each construct type.Alternatively the first construct type can be introduced into a firstplant while the second construct type can be introduced into a secondisogenic plant, following which the transgenic plants resultanttherefrom can be crossed and the progeny selected for doubletransformants. Further self-crosses of such progeny can be employed togenerate lines homozygous for both constructs.

As mentioned the components of the systems of the present invention mayalso be combined in non-cellular particles or reactors.

As used herein, the term “combining” refers to any method where thecomponents are in close enough proximity that carbon fixation may occur.Thus, the term “combining” incorporates such methods as co-expressingand co-solubilizing the components of the present invention.

It will be appreciated that in a non-cellular system the components ofthe carbon fixation pathway are typically expressed in host cells andfollowing a predetermined time in culture, recovery of the recombinantpolypeptide (enzyme) is effected.

The phrase “recovering the recombinant polypeptide” used herein refersto collecting the whole fermentation medium containing the polypeptideand need not imply additional steps of separation or purification.

Thus, polypeptides of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, salting out (as in ammonium sulfate precipitation), affinitychromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

To facilitate recovery, the expressed coding sequence can be engineeredto encode the polypeptide of the present invention and fused cleavablemoiety. Such a fusion protein can be designed so that the polypeptidecan be readily isolated by affinity chromatography; e.g., byimmobilization on a column specific for the cleavable moiety. Where acleavage site is engineered between the polypeptide and the cleavablemoiety, the polypeptide can be released from the chromatographic columnby treatment with an appropriate enzyme or agent that specificallycleaves the fusion protein at this site [e.g., see Booth et al.,Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem.265:15854-15859 (1990)].

The polypeptide of the present invention is preferably retrieved in“substantially pure” form.

As used herein, the phrase “substantially pure” refers to a purity thatallows for the effective use of the protein in the applicationsdescribed herein.

In addition to being synthesizable in host cells, the polypeptide of thepresent invention can also be synthesized using in vitro expressionsystems. These methods are well known in the art and the components ofthe system are commercially available.

As mentioned, to support carbon fixation in a non-cellular system, theenzymatic system should be provided with energized cofactors, namely ATPand NADH and/or NADPH. These cofactors can be regenerated in vitro invarious ways (Wichmann R & Vasic-Racki D (2005) (SpringerBerlin/Heidelberg), Vol 92, pp 225-260).

In one embodiment, the pathway components of the present invention arecombined in a carrier system (i.e., encapsulating agent) of desiredproperties. In a specific embodiment, the encapsulating agent is aliposome.

As used herein and as recognized in the art, the term “liposome” refersto a synthetic (i.e., not naturally occurring) structure composed oflipid bilayers, which enclose a volume. Exemplary liposomes include, butare not limited to emulsions, foams, micelles, insoluble monolayers,liquid crystals, phospholipid dispersions, lamellar layers and the like.The liposomes may be prepared by any of the known methods in the art[Monkkonen, J. et al., 1994, J. Drug Target, 2:299-308; Monkkonen, J. etal., 1993, Calcif. Tissue Int., 53:139-145; Lasic D D., LiposomesTechnology Inc., Elsevier, 1993, 63-105. (chapter 3); Winterhalter M,Lasic D D, Chem Phys Lipids, 1993 September; 64(1-3):35-43]. Theliposomes may be positively charged, neutral, or, negatively charged.

The liposomes may be a single lipid layer or may be multilamellar.Surfactant peptide micelles are also contemplated.

In another embodiment, the pathway components of the present inventionare embedded in a carrier (i.e., embedding agent) of desired properties.In specific embodiments, the embedding agent (or carrier) is amicroparticle, nanoparticle, nanosphere, microsphere, nano-plate,microcapsule, or nanocapsule [M. Donbrow in: Microencapsulation andNanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton, Fla.,347, 1991]. The term carrier includes both polymeric and non-polymericpreparations. According to a specific embodiment, the embedding agent isa nanoparticle. The polypeptides of the present invention may beembedded in the nanoparticle, dispersed uniformly or non-uniformly inthe polymer matrix, adsorbed on the surface, or in combination of any ofthese forms. Polymers which may be used for fabricating thenanoparticles include, but are not limited to, PLA (polylactic acid),and their copolymers, polyanhydrides, polyalkyl-cyanoacrylates (such aspolyisobutylcyanoacrylate), polyethyleneglycols, polyethyleneoxides andtheir derivatives, chitosan, albumin, gelatin and the like.

It will be appreciated that the enzymes of the present invention and theelectron donor need not be encapsulated. Thus, according to yet anotherembodiment, the enzymes and the electron donor of the present inventionare free in solution.

In yet embodiment, the pathway components of the present invention arecombined in a reactor of desired properties. Exemplary reactors include,but are not limited to a test-tube, a container, a bioreactor and avessel.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalor calculated support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Pathway Analysis Metrics Enable a Comprehensive ComparisonBetween Pathways

Many different aspects of a given metabolic pathway are important forits function. To enable evaluation and comparison of metabolic pathways,the present inventors have used several parallel criteria.

The pathway specific activity (criterion I) is analogous to an enzyme'sspecific activity and is defined to be the maximal rate of productformation by 1 mg of pathway total protein (see Example 2 for the exactcalculation). The pathway specific activities for all natural carbonfixation pathways have been calculated and are presented in FIG. 12,Table 1 (marked in blue).

These pathways, as well as the other that were analyzed in Example 2(including the natural pathways) are annotated and grouped according totheir main metabolic characteristics, i.e., the carboxylating enzymesthat create their “metabolic core”. Aside from those enzymes, a pathwayis generally annotated according to an indicative metabolite that doesnot participate in other pathways sharing the same carboxylatingenzymes. For example, the MOG pathways (FIG. 8) are marked as PEPC,PyrC, PEPC-AcC or PyrC-AcC depending on the carboxylating enzymes usedby the specific MOG variant. The indicative metabolites are alanine orlactate, depending on the ‘bypass’ taken (module C in FIG. 8).

Table 1 (FIG. 12) is divided according to several criteria. The maindivision is between ferredoxin-oxidoreductase-containing andnon-ferredoxin-oxidoreductase pathways. The ferredoxin-oxidoreductaseenzymes present two main difficulties: they are extremely oxygensensitive and the rate of carboxylation they support is generallyun-known. Because one cannot obtain reliable information regarding theirenzymatic rates they were not included in the pathway specificactivities calculation. Therefore, the pathways containing those enzymeshave an artificially higher specific activity and cannot be compared tothe non-ferredoxin-oxidoreductase-containing pathways.

The natural pathways are given at the beginning of each section and aremarked in blue. Throughout the text and in Table 1, FIG. 12 thefollowing abbreviations for the carboxylating enzymes are used: AcC:acetyl-CoA carboxylase; CCR: crotonyl-CoA carboxylase/reductase; ICDH:isocitrate dehydrogenase; KGC: 2-ketoglutarate carboxylase; KGS:2-ketoglutarate synthase; ME: ‘malic’ enzyme; MCC: methylcrotonyl-CoAcarboxylase; PEPC: PEP carboxylase; PrC: propionyl-CoA carboxylase;PyrC: pyruvate carboxylase and PyrS: pyruvate synthase.

The ubiquitous rPP cycle has a pathway specific activity of 0.25μmole/min/mg (taking into account oxygenase activity), a value that willbe used as a benchmark for evaluation of the kinetics of the syntheticpathways (the conditions under which this criterion reflects the pathwayflux are detailed and elaborated in Example 2 herein below).

While the kinetics of a pathway is of central importance it does notprovide any information regarding the cellular resources it consumes.Different pathways, which ultimately perform the same metabolicconversation, might consume the different resources of the cell to adifferent measure, thereby unequally affecting the organism growth. Thepresent inventors focus on the energetic cost (criterion II) associatedwith the different pathways, corresponding to the efficiency of usingthe light-regenerated resources of the cell. The energetic cost can beseparated into two terms:

NADPH cost: the number of moles of NADPH equivalents (i.e. redoxcarriers, such as NAD(P)H, ferredoxins and FADH₂) consumed in theproduction of one mole of product (GA3P).

ATP cost: The number of moles of ATP equivalents (non-redox energycarriers, e.g. NTPs, phosphate esters and coenzyme A thioesters)consumed in the production of one mole of product.

The NADPH and ATP costs of all natural carbon fixation pathways aregiven in FIG. 12, Table 1.

The energetic cost of a pathway can be used to determine the energeticfeasibility of the pathway as a whole as well as any of its parts. Athermodynamicaly favorable (criterion III) pathway is one for which thefree energy change (ΔG^(r)) associated with the production of one moleof product is negative. A negative free energy change is also requiredto be obtained for each part of the pathway under physiological range ofmetabolite concentrations (See Example 2).

In order to ensure thermodynamic feasibility, a carbon fixation pathwaymust involve the hydrolysis of a certain minimal amount of ATPmolecules. Yet, hydrolysis of too many ATP molecules will decrease theenergetic efficiency (increase the energetic cost) considerably. Thisminimal ATP requirement depends on the identity of the differentelectron donors utilized by the pathway, on the pH and on the ionicstrength (Alberty R A (2003) Thermodynamics of Biochemical Reactions(Wiley-Interscience)). In FIG. 2A, the minimal ATP requirement of theprevalent rPP pathway in physiological ionic strengths and pH values isanalyzed, where all the electron donors are NAD(P)H and under ambientCO₂ ^(gas) concentration of ˜390 ppm. As the figure shows, 5-6 ATPs areminimally required (see Example 2). It may be further noted that the rPPuses 9 ATPs in order to support carbon fixation under ambientconditions; the extra ATP molecules consumed are suggested to enhanceits kinetics at the expense of its ATP efficiency.

Different electron donors, other than NAD(P)H, with lower (i.e. moreenergetic, e.g. ferredoxin) or higher (e.g. menaquinone) reductionpotentials would shift this thermodynamic profile (Example 2). FIGS. 2Band C show the minimal ATP requirement for the rAcCoA pathway and therTCA cycle, which utilize these electron donors. Interestingly, althoughthese pathways are potentially more efficient (hydrolyzing only 4-5ATPs), it may be concluded that they are not feasible at pH valueshigher than 7 under ambient CO₂ concentrations (bold lines correspond tothe feasibility ranges in FIG. 2). These results are in agreement withthe observation that organisms that operate the rTCA cycle or the rAcCOApathway usually occupy high CO₂ habitats or operate a carbonconcentrating mechanism (FIG. 10B). These organisms are generallyanaerobic and energy restricted as compared to aerobes, which limitstheir available energy for investment in carbon fixation. Hence, theyprefer the utilization the rTCA cycle or the rAcCOA pathway that aremore ATP efficient than the rPP cycle.

While the kinetics and the energetics of a pathway provide valuableinformation concerning its function, there are several other factorsthat can further aid in the assessment and comparison of the differentmetabolic alternatives. Of these, the present inventors addressed thetopology (criterion IV), corresponding to the internal makeup of apathway and its integration with the structure of the metabolic networkof the cell. The topology criterion incorporates two importantparameters:

Number of enzymes (simplicity): The number of enzymes the carbonfixation cycle utilizes as an independent unit, as well as the number ofenzymes the complete pathway employs (including the conversion of thecycle's product into triose-phosphate).

Metabolic compatibility of the synthetic pathways: In designingalternative CO₂ assimilation pathways, it is important to consider howthe pathways will integrate into the endogenous metabolic network. Thepresent inventors have used a model of central carbon metabolism in thealgae Chlamydomonas and employed constrained-based analysis (i.e., fluxbalance analysis (FBA) and uniform random sampling (Schellenberger J &Palsson B O (2009) J Biol Chem 284(9):5457-5461)) to test thecompatibility of each cycle with the endogenous metabolic network (LewisN E, et al (2009) Metabolic Systems Biology: A Constraint-BasedApproach. Encyclopedia of Complexity and Systems Science). The presentinventors have calculated the growth yield supported by each pathway aswell as the number of significantly changed fluxes in the modifiednetwork as compared to the wild-type model (See Example 2).

A Systematic Method to Locate Novel Synthetic Carbon Fixation PathwaysReveals the Simplest Carbon Fixation Cycles

The present inventors have developed a novel computational approach (seeExample 2) to systematically explore all the possibilities to buildcarbon fixation cycles of a given size that utilize the ˜4000 enzymesreported in the KEGG database. Each candidate cycle employs one or morecarboxylating enzymes and produces an organic output compound with atleast two carbons. This exhaustive search enables covering a space ofpossibilities which was next analyzed for its feasibility andfunctionality using the criteria detailed above.

The analysis was started by exploring what are the synthetic carbonfixation pathways which employ the simplest (shortest) cycles. Suchpathways can be a priori suggested to be attractive in terms of rate.Several pathways were found that employ cycles with merely four to sixenzymatic steps (FIGS. 7A-G). FIG. 3 presents one of the three cyclesthat utilizes only four enzymes. This cycle is a metabolic shortcut ofthe naturally occurring rTCA cycle; three of its enzymes participate inthe rTCA cycle and the enzyme isocitrate lyase metabolically bypassesthe rest of the natural cycle. The product of this simple cycle,glyoxylate, is converted to GA3P by the bacterial-like glycerate pathway(Kebeish R, et al. (2007), Nat Biotechnol 25(5):593-599) (FIG. 6).

However, this as well as the other pathways that employ such ultra-shortcycles, suffer from a fundamental fault. Most cycles, as distinctmetabolic units that produce glyoxylate, are not thermodynamicallyfeasible, taking into account the physiologically relevant glyoxylateconcentrations (see Example 2). In addition, all use an oxygen-sensitiveferredoxin-oxidoreductase enzyme (pyruvate synthase or 2-ketoglutaratesynthase) and have a significantly lower pathway specific activity, ascompared to the rTCA cycle (see FIG. 12, Table 1).

Novel Kinetically Efficient Carbon Fixing Pathways Utilizing the MostAttractive Carboxylating Enzymes

The design of kinetically efficient pathways requires the utilization ofhigh carboxylating enzymes, which result from having high specificactivities and affinities towards CO₂ or HCO₃ ⁻. A wide literaturesurvey was performed and kinetic properties of known carboxylatingenzymes were compared as presented in FIG. 13, Table 2.

Phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase are themost favorable carboxylating enzymes; both have high specific activitiesand superior affinity for HCO₃ ⁻. Acetyl-CoA and Propionyl-CoAcarboxylases are the next favorable. These four enzymes prefercarboxylation over decarboxylation. Isocitrate dehydrogenase, whichprefers decarboxylation under common physiological conditions, is also akinetically acceptable option. All other carboxylating enzymes arerather slow under ambient CO₂/HCO₃ ⁻ concentrations (specific activity<2 μmol/min/mg).

The present inventors used a systematic search tool to find the shortestpathways that employ different sets of the favorable carboxylatingenzymes (Example 2). The present inventors have numerically predictedwhich of these pathways is best in terms of pathway specific activity.Notably it was found that all the pathways with the highest specificactivities employ similar cycles, with a shared metabolic core structure(FIG. 8). The product of those cycles, glyoxylate, is assimilated by thebacterial-like glycerate pathway. This family of pathways has beentermed the Malonyl-CoA-Oxaloacetate-Glyoxylate (MOG) pathways. It hasbeen found that the MOG pathways have 2-3 fold higher pathway specificactivity as compared to the rPP cycle (FIG. 12, Table 1). FIGS. 4A-Bpresent two MOG pathways that employ only one carboxylating enzyme, thesuperior PEP carboxylase. These MOG pathways have been termed theC4-Glyoxylate cycles, because they converge with the naturally evolvedC4 mechanism (FIG. 4C).

In C4 plants carbon is temporarily fixed, in the mesophyll cells, by thecarboxylation of PEP to oxaloacetate. This is followed by the reductionof oxaloacetate to malate. Malate is then transported to thebundle-sheath cells where it releases the CO₂, which is re-assimilatedby Rubisco. Pyruvate is recycled to complete the cycle which serves as a“futile cycle” that concentrates CO₂ (Nelson D L & Cox M M (2004)Lehninger Principles of Biochemistry (W. H. Freeman & Co.). All of thesereactions, with the exception of decarboxylation, appear in theC4-Glyoxylate cycle. Therefore, the C4-Glyoxylate cycles are analternative for completing the C4 cycle without “losing” the carbon:replacing the “futile” decarboxylation reaction with an extracarboxylation, accompanied with the export of glyoxylate.

There are several possible variations on the C4-Glyoxylate cycles (FIG.8) each having possible pros and cons. Using Pyruvate carboxylaseinstead of PEP carboxylase results in higher ATP efficiency but somewhatlower pathway specific activity (FIG. 12, Table 1). In addition,acetyl-CoA carboxylase, another efficient carboxylating enzyme, can beused instead of the carboxytransferase enzyme (denoted as #6 in FIG. 4).FIG. 12, Table 1 presents a comprehensive comparison between all the MOGpathways.

The MOG pathways are equivalent to the rPP cycle in their electrondonors usage; all donors are NAD(P)H. Hence, FIG. 2A presents the ATPrequirement for the MOG pathways as well. These pathways hydrolyze 8-12ATP molecules (depending on the exact pathway identity, see Example 2)and therefore are all thermodynamically feasible, with ΔG<<0 under awide range of pH and ionic strengths.

Using the central carbon metabolism model of the algae Chlamydomonas itwas found that the MOG pathways were able to support maximal growthyield with no further secretion products. Moreover, the flux solutionspace indicates that the integration of the MOG cycles necessitates thefewest significant changes in the endogenous flux distributions incomparison to. (Example 2).

Most reactions employed by the MOG pathways are prevalent in manyspecies throughout the tree of life. Yet, some reactions involved inthese pathways are rather unique. For example, the reduction ofmalonyl-CoA (reaction 7 in FIGS. 4A and B) can be performed by an enzymefound only from thermophilic prokaryotes (Alber B, et al. (2006), JBacteriol 188(24):8551-8559). In addition, the hydration of acrylyl-CoA(reaction 10 in FIG. 4B) is carried out by the enzyme lactoyl-CoAdehydratase that contains iron-sulfur centers and was found to be oxygensensitive (Kuchta R D & Abeles R H (1985) J Biol Chem260(24):13181-13189). Nevertheless, early studies indicate that thereexist variants of the enzyme, from various organisms, that showefficient performance under full aerobic conditions (e.g. Baldwin R L,Wood W A, & Emery R S (1965) Biochim Biophys Acta 97:202-213)). Finally,the enzyme alanine aminomutase (reaction 9 in FIG. 4A) was evolved fromthe enzyme lysine 2,3-aminomutase to act on alanine (Liao H H, Gokarn RR, Gort S J, Jessen H J, & Selifonova O (2007) U.S. Pat. No. 7,309,597B2.). A comprehensive discussion on the unique enzymes of theC4-Glyoxylate cycles is given in Example 2.

Other promising synthetic carbon fixation pathways that resulted fromthe present analysis are discussed in Example 2 (FIGS. 9A-N and FIG. 12,Table 1). Notably, many of the synthetic pathways are expected to befaster than the rPP cycle (i.e., they have higher calculated pathwayspecific activities).

Analysis and Optimization of Carbon Fixation Pathways

This study used a novel methodology to computationally analyze andcompare carbon fixation pathways, by focusing on their kinetics. It wasfound that synthetic pathways have the potential to show significantlyfaster kinetics as evaluated by the pathway specific activity. From abiotechnological point of view, this criterion is probably a major one,directly affecting the productivity of a photosynthetic, carbon fixingorganism. Importantly, under ambient conditions and averageillumination, the ATP and NADPH costs are suggested to rarely be alimiting factor (Holt N E, Fleming G R, & Niyogi K K (2004) Biochemistry43(26):8281-8289). As shown in the present analysis the NADPH costs ofall natural and synthetic pathways are the same (FIG. 12, Table 1),corresponding to the fact that the same number of electrons is requiredto reduce inorganic carbon to GA3P regardless of the exact metabolicpath. Notably, the NADPH cost of the rPP cycle, which includesphotorespiration, is higher because some of the electrons are used byglycolate oxidase to reduce molecular oxygen into H₂O₂.

The overall flux through a pathway is approximated by the pathwayspecific activity criterion when: (1) the enzymes are substratesaturated, (2) the rate of the backward reaction of each enzyme isnegligible compared to the rate of its forward reaction and (3) enzymeexpression levels are balanced based on each enzyme's specificactivities (no ‘surplus’ of any enzyme). Obviously, in natural pathwaysnone of these requirements fully holds; therefore the pathway specificactivity serves as an upper limit estimation of the pathway overall rate(Example 2). It is used only as a useful, well-defined proxy whichenables calculation and comparison with the limited available kineticdata. This metric is not biased and thus the advantage of the syntheticalternatives over the natural pathways is expected to hold even if theoverall rates would be lower than predicted.

Several other optimization methods have been discussed in theliterature, based on minimization of overall metabolic intermediateconcentration, minimization of transient times, and maximization ofenzyme specificity (Heinrich R, Schuster S, & Holzhutter H G (1991) EurJ Biochem 201(1):1-21). However, most of these cannot be systematicallyemployed due to the lack of necessary data.

In this study, the present inventors have referred explicitly only tothe efficiency of using ATP-like and NADPH-like resources, bothregenerated by light. In reality, however, the water usage efficiency,the nitrogen usage efficiency and others are just as important even inhuman cultivated environment. It is important to note, however, thatutilizing a carbon fixation pathway with increased productivity isexpected to have a considerable positive effect on these efficiencies aswell. For example, higher specific rate of carbon fixation will enablethe cell to better reallocate its resources, e.g. dedicate less proteinfor carbon fixation, which in turn will increase the nitrogen useefficiency (photosynthetic rate per unit of N, [Sage R F & Pearcy R W(1987) Plant Physiol 84(3):959-963). In addition, the increased affinitytowards inorganic carbon and the absence of the oxygenation reactionwill enable the plant to sustain a high carbon fixation rate even when ahigh fraction of the pores are closed, which in turn will increase thewater use efficiency.

Example 2

In the present example a comprehensive view of the synthetic carbonpathways discovered using the described search is provided.

General Aspects of the Synthetic Carbon Fixation Pathways

To enable pathway evaluation and comparison in terms of the differentcriteria, a common pathway product was defined.Glyceraldehyde-3-phosphate (GA3P) was selected as such a metabolitebecause it is regarded as the product of the reductive pentose phosphate(rPP) cycle (FIG. 5A) and because it is the simplest sugar leading tothe bio-synthesis of larger transport metabolites. Each pathway istherefore composed of a cycle and an assimilation sub-pathway whichconverts the cycle's product into GA3P (FIG. 6). The choice of whichcompound will serve as the pathway output does not affect thequalitative results discussed here. It may offset the energetic cost orthe pathway specific activity but to similar amounts in all pathways. Itis thus a useful approach but one that can be changed to a differentchoice without invalidating the present conclusions.

To ensure the correct forward direction of the metabolic flux throughthe different pathways, it is important for them to utilize at least oneirreversible enzyme. Importantly, all the synthetic pathways proposed inthe present examples contain this feature. Such irreversible reactionsinclude PEP carboxylation by PEP carboxylase (as opposed to reversiblePEP carboxylation by PEP carboxykinase), glyoxylate self-condensation(forming tartronate-semialdehyde), glycerate phosphorylation, malatedecarboxylation and propionyl-CoA formation (acrylyl-CoA reduction).

Importantly, almost none of the proposed cycles are auto-catalytic, asthe rPP cycle (the product of an auto-catalytic cycle is also anintermediate of the cycle; as glyceraldehydes-3-phosphate in the case ofthe rPP cycle). Therefore, the proposed cycles avoid complex regulationthat must be imposed in order to maintain appropriate metaboliteconcentrations in auto-catalytic cycles.

Many proposed synthetic cycles utilize the enzyme PEP carboxylase. PEPcarboxylase from C4 plants is known to be light regulated and thereforecan serve in switching the cycle activity according to the lightexposure.

II. The MOG Pathways: Characteristics and Unique Reactions

A Group of Pathways that have the Highest Calculated Pathway SpecificActivity

The present search for synthetic carbon fixation cycles revealed apromising group of pathways. The basic structure of the pathways thatbelong to this group is shown in FIG. 8. Malonyl-CoA and oxaloacetateare the products of the pathways' carboxylation or transcarboxylationreactions and glyoxylate is the common export product of these pathways.Therefore, this pathway group has been termed theMalonyl-CoA-Oxaloacetate-Glyoxylate (MOG) pathways.

The MOG pathways have the highest calculated pathway specific activitiesand thus show promise to have the highest rates of carbon fixation amongthe synthetic carbon fixation pathways. The reason for their suggestedsuperiority is the unique combination of the carboxylating enzymes theyutilize. The MOG pathways utilize only the three best carboxylatingenzymes: PEP, pyruvate and acetyl-CoA carboxylase. Those three enzymesare characterized by high specific activities under saturating CO₂/HCO₃⁻ concentrations and by excellent affinities for HCO₃ ⁻, which, in turn,give them the highest specific activities, under ambient CO₂/HCO₃ ⁻concentrations, in comparison to all other carboxylating enzymes (seeFIG. 13, Table 2).

As shown in FIG. 8, different MOG pathways employ different combinationsof these superior carboxylating enzymes. Pyruvate carboxylase canreplace PEP carboxylase (options I and II of module C), which results ina decrease in the ATP cost, but also in the pathway specific activity(FIG. 12, Table 1, PyrC and Pyr-AcC, marked in green). Acetyl-CoAcarboxylase can be replaced by a transcarboxylase enzyme, coupled withan extra carboxylation of pyruvate/PEP to oxaloacetate (options I and IIof module A). In addition, the metabolic conversion of malonyl-CoA topyruvate can be accomplished by different “bypasses”, such as the“alanine bypass” (FIG. 4A) or the “lactate bypass” (FIG. 4B). FIG. 12,Table 1 presents the pathway specific activities calculated for each ofthose combinations and demonstrate that all of them are expected to beexcellent carbon fixation pathways.

FIG. 4 shows the MOG pathways that use PEP carboxylase as their solecarboxylating enzyme. The main advantage of these pathways over theircounterparts is having the highest predicted pathway specific activity(FIG. 12, Table 1). They can also have advantages in terms ofregulation. PEP carboxylase from C4 plants is known to be lightregulated, a mechanism that can be used in the host organism to shut thecycle down when illumination falls below a certain threshold. The lightactivation of the other C4 enzymes presented in the cycle (pyruvatedikinase and malate dehydrogenase), by similar mechanisms, can supportthis kind of regulation.

Below reactions which are specific to the ‘lactate’ and ‘alanine’bypasses of the MOG pathways are discussed.

Unique Reactions of the “Alanine Bypass”

The “alanine bypass”, converting malonyl-CoA to pyruvate, is shown inFIG. 8, module B, option 2. The two unique reactions in this pathway arethe reduction of malonyl-CoA to malonate-semialdehyde and theaminomutase reaction that converts beta-alanine to alpha-alanine.

Two archaeal strains, Sulfolobus tokodaii and Metallosphaera sedula,were found to employ a unique malonyl-CoA reductase enzyme thatcatalyzes the reduction of malonyl-CoA to malonate-semialdehyde(reaction 7 in FIG. 4A) (Alber B, et al. (2006) J Bacteriol188(24):8551-8559).

The enzyme alanine aminomutase (reaction 8 in FIG. 4A) reversiblyconverts beta-alanine to alpha-alanine. This enzyme was evolved from theenzyme lysine 2,3-aminomutase to act on alanine (Prather K L & Martin CH (2008) Curr Opin Biotechnol 19(5):468-474; Liao H H, et al (2007) U.S.Pat. No. 7,309,597 B2). Importantly, the Bacillus subtilis lysine2,3-aminomutase was chosen for the cloning and the screening processes.This enzyme, unlike several others that exist in other organisms, isstable in air and remains fully active under aerobic conditions.

Unique Reactions of the “Lactate Bypass”

The “lactate bypass”, converting malonyl-CoA to pyruvate, is shown inFIG. 8, module B, option 1. The unique reactions in this pathway aredescribed below:

The reduction of malonyl-CoA to 3-hydroxypropionate (reaction 7 in FIG.4B) can be performed by the enzyme malonyl-CoA reductase from the greennonsulfur bacterium Chloroflexus aurantiacus, which was found tocatalyze the two-step reduction of malonyl-CoA to 3-hydroxypropionate(Hugler et al, 2002, J Bacteriol 184(9):2404-2410). This enzyme containstwo separate domains for the two step reaction: an aldehydedehydrogenase and an alcohol dehydrogenase. Malonate-semialdehyde, theproduct of the first reaction, serves as a soluble, free substrate forthe second reaction. The optimum temperature of this enzyme is >50° C.

Another important enzyme utilized by the lactate bypass is propionateCoA transferase (EC 2.8.3.1) (reaction 8 in FIG. 4B). This enzyme isknown to catalyze a CoA transfer reaction between acetyl-CoA andpropionate or between propionyl-CoA and acetate. However, the enzyme canalso accept 3-hydroxypropionate and lactate as substrates. Therefore,the present inventors have integrated this enzyme in the proposedlactate bypass, to catalyze the CoA transfer between (R)-lactoyl-CoA and3-hydroxypropionate (reaction #8). To estimate the specific activity ofthis reaction the present inventors have assumed it involves two steps:CoA transfer from (R)-lactoyl-CoA to acetate and a CoA transfer fromacetyl-CoA to hydroxypropionate. Monitoring the rate of CoA transferfrom acetyl-CoA, (R)-Lactate is a better substrate than acetate andalmost as good as propionate. The rate in which the enzyme catalyzes theCoA transfer between acetyl-CoA and 3-hydroxypropionate is much slower:only 32% of the CoA units were transferred to 3-hydroxypropionate fromacetyl-CoA (as compared to 80% for propionate, 65% for lactate and 60%for acrylate).

The hydratase enzyme crotonase (enoyl-CoA hydratase, EC 4.2.1.17)catalyzes the reversible hydration of crotonyl-CoA and of long chain(trans) 2,3-unsaturated fatty acids. The enzyme can also catalyze thereversible hydration of acrylyl-CoA to 3-hydroxypropionyl-CoA (reaction9 in FIG. 4B). However, the specific activity of the enzyme for thisreaction was not tested. Yet, the enzyme displays an extremely highspecific activity for crotonyl-CoA hydration and hence even taking anextremely conservative estimation gives a significant activity.

The hydration of acrylyl-CoA to lactoyl-CoA is catalyzed by lactoyl-CoAdehydratase (EC 4.2.1.54) (reaction 10 in FIG. 4B). The enzyme, purifiedfrom the anaerobic prokaryote Clostridium propionicum, was extensivelystudied and was found to contain iron-sulfur centers and to be extremelyoxygen sensitive: after a 1-min exposure to air, activity is inhibitedby more than 90%. However, studies of lactoyl-CoA dehydratase from otherorganisms, such as Pseudomonas sp., Peptostreptococcus elsdenii andpigeon liver, showed efficient performance under full aerobicconditions.

III. The Simplest Carbon Fixation Cycles

The present search has found several ultra-short cycles that are able tofix carbon. Those cycles are shown in FIGS. 7A-G. All of these cyclesutilize one ferredoxin-oxidoreductase enzyme. Interestingly, they allcan be regarded as “metabolic shortcuts” of the reductive TCA cycle; theKGS-ICDH and the KGS-KGC pathways (FIGS. 7A-B) share all theirmetabolites with the rTCA cycle and use the enzyme isocitrate lyase as a“metabolic bridge” and thus fix two CO₂ molecules to form glyoxylate,which in turn can be converted into GA3P by the bacterial-like glyceratepathway. In addition, the PyrS-ME, PyrS-PyrC and the PyrS-PEPC pathways(FIGS. 7C-E) share their metabolites with the rTCA cycle, with malyl-CoAas the only foreign metabolite. The PyrS-PyrC-Oxalate and thePyrS-PEPC-Oxalate pathways (FIGS. 7F-G) are somewhat furthermetabolically distant from the rTCA cycle, but still share with it majormetabolites.

The most attractive pathway of this group, PyS-PEPC-Glyoxylate, is acombination of the metabolic routes B and E, shown in FIG. 1. Thoseroutes share three enzymes that can be discarded to create an efficient“metabolic shortcut” that produces glyoxylate, which is then assimilatedby the bacterial-like glycerate pathway. This cycle was previouslypostulated, and named the reductive dicarboxylic acid cycle, butevidence for its natural existence is scarce. The pathway isthermodynamically feasible, does not contain a thermodynamic distributedbottleneck (see below) and is energy efficient. However, consistent withall the other pathways that use simple cycles, its pathway specificactivity is significantly lower than that of the naturalferredoxin-oxidoreductase pathways, the rTCA cycle and the DC/4-HBcycles.

Out of the three pathways that use a 4-enzyme-cycle, two (KGS-ICDH andPyrS-ME) are not thermodynamically feasible because they contain athermodynamic distributed bottleneck; the free energy change associatedwith their cycles is positive under the reasonable range of estimatedphysiological concentrations of their substrates and products (Table 1,FIG. 12). In addition, the “malic” enzyme (of the cycle PyrS-ME)strongly prefers decarboxylation under ambient CO₂ concentration. Usingthis enzyme for carboxylation is highly questionable.

Two of the pathways that use a 5-enzymes-cycle (KGS-KGC andPyrS-PyrC-Glyoxylate) are thermodynamically questionable. This isbecause the free energy change associated with their cycles may bepositive under a broad range of estimated physiological concentrationsof their product (FIG. 12, Table 1). Moreover, the KGS-KGC cycleutilizes the enzyme 2-ketoglutarate carboxylase that was found tooperate only in one thermophilic bacterium, Hydrogenobacterthermophilus, with an optimum temperature of 70-80° C. This makes thecycle even less attractive. The PyrS-PyrC-Oxalate and thePyrS-PEPC-Oxalate cycles (FIGS. 7F-G) are thermodynamically feasible buttheir energy efficiency is quite low (ATP cost of 10 and 12,respectively). To conclude, none of the simplest cycles present apromising alternative to the natural pathways. Simplicity alone does notseem to be a good indicator of the usefulness of a pathway, in terms ofrate (the natural, complex rTCA and 3-HP/4-HB cycles have significantlyhigher specific activities), of energetic balance and of the enzymesemployed (all use ferredoxin-oxidoredcutase enzymes).

IV. Other Interesting Carbon Fixation Pathways

The AcC-ICDH Cycles

An interesting enzyme, 2-hydroxyglutarate synthase (EC 2.3.3.11), foundto operate in Escherichia coli, has potential use in synthetic pathwaydesign. Two efficient carbon fixation pathways can be suggested by usingits catalytic condensation of propionyl-CoA and glyoxylate (FIGS. 9A-C).Both use the carboxylating enzymes acetyl-CoA carboxylase and isocitratedehydrogenase, for which the V_(max) for carboxylation, under ambientCO₂ concentration, are rather high (FIG. 13, Table 2, and Example 1).

The first pathway (FIG. 9A), the AcC-ICDH-Glycerate cycle, producesglyoxylate that can be assimilated using the bacterial-like glyceratepathway or by the “citramalate” glyoxylate assimilation pathway (FIG.6). The former option was found to have a higher pathway specificactivity and is analyzed in FIG. 13, Table 2.

The second pathway (FIG. 9B), the AcC-ICDH-Pyruvate cycle, generatesacetyl-CoA. Acetyl-CoA can then be assimilated to pyruvate using thesub-pathway shown in Aiii. Pyruvate is converted to GASP by thegluconeogenesis.

In terms of pathway specific activity, both pathways are significantlyfaster than the rPP cycle (at 20% oxygenase reaction of RUBISCO) and the3-HP pathway. The AcC-ICDH-Glycerate pathway is simpler and more ATPefficient.

The AcC-PrC Cycles

The naturally found 3-HP pathway creates the basic structure of a cyclethat uses acetyl-CoA and propionyl-CoA carboxylases as solecarboxylating enzymes. It is actually composed of two sequential cycles,with glyoxylate as the product of the first one. As a “shortcut”alternative to the second cycle (the “citramalate cycle”), whichcondenses glyoxylate with propionyl-CoA to produce acetyl-CoA andpyruvate, glyoxylate can be condensed by the bacterial-like glyceratepathway to produce glycerate (FIG. 6, FIG. 9D). This option waspreviously considered and was not found to function in the organismstested (Herter S, et al. (2001) J Bacteriol 183(14):4305-4316). One ofthe reasons for the dismissal of this alternative was that thebacterial-like glycerate pathway involves the release of a CO₂ molecule(FIG. 6), which seems to counteract the carbon fixation role of thepathway (Herter S, et al. (2001) J Bacteriol 183(14):4305-4316).However, the present rate analysis demonstrates that theAcC-PrC-Glycerate pathway is not only more ATP efficient and simpler,but also significantly faster than the naturally foundAcC-PrC-Citramalate pathway, even when taking into account the CO₂released (Table 1, FIG. 12). This indicates that contrary to intuitivethinking, the AcC-PrC-Glycerate pathway might serve as an excellentcarbon fixation pathway.

FIGS. 9D-G illustrate another derivative of the 3-hydroxypropionatepathway, the AcC-PrC-Citrate pathway. The cycle produces acetyl-CoA(FIG. 9E) which can be assimilated into pyruvate using a metabolicderivative of the main cycle (FIG. 9F). Pyruvate is converted to GA3P bygluconeogenesis. As shown in, the pathway is as fast and as ATPefficient as the other AcC-PrC pathways.

A non-ferredoxin-oxidoreductase-containing pathway can be derived fromthe 3-HP/4-HB pathway, by converting the cycle's intermediate,succinyl-CoA, to malate and then to pyruvate (FIG. 9G). The pathway hasa comparable rate to the 3-HP/CM pathway. However, the pathway utilizestwo oxygen sensitive enzymes, 4-hydroxybutaryl-CoA dehydrates andsuccinate-semialdehyde dehydrogenase, which decrease its overallusability.

The Crotonyl-CoA Cycles

Three interesting cycles can be constructed around crotonyl-CoA (FIGS.9H-J). Those cycles utilize different sets of carboxylating enzymes.

FIG. 9H displays an interesting pathway that uses a newly discoveredunusual carboxylating enzyme, crotonyl-CoA carboxylase-reductase.However, the CCR-PEPC pathway is rather slow and is characterized by aspecific activity comparable to that of the rPP cycle.

FIGS. 9I-J present carbon fixation pathways that utilize the enzymemethylcrotonyl-CoA carboxylase. Glyoxylate, produced by the cycles isassimilated by the bacterial-like glycerate pathway. The MCC-ICDHpathways are the slowest pathways analyzed. This is because thecarboxylation of crotonyl-CoA is relatively inefficient (FIG. 13, Table2). Moreover, the MCC-ICDH pathways utilize the oxygen sensitive enzyme2-hydroxyglutaryl-CoA dehydratase, which further decreases the overallusability of the pathways. The MCC-ICDH-4HB cycle uses other oxygensensitive enzymes that participate in the natural 3-HP/4-HB cycle.

The AcC-PrC-KGS-ICDH Cycle

FIG. 1, which displays the naturally occurring carbon fixation pathways,hides another uncommon combination of metabolic routes D and A thatgenerate a metabolic hybrid of the 3-hydroxypropionate pathway and therTCA cycle. However, the AcC-PrC-KGS-ICDH pathway is not an efficientone. Its pathway specific activity is only 0.67 mol/min/mg, which ismuch lower than that of the other ferredoxin-oxidoreductase pathways.

The Glutamate Cycles

Four interesting pathways can be constructed using a mutase enzyme thatconverts glutamate to methylaspartate (FIGS. 9K-M). The first two areshown in FIG. 9K. The two left “downward” metabolic routes correspond tothe naturally occurring routes B and D of FIG. 1. ThePyrS-PEPC-KGS-Glutamate pathway has a pathway specific activity that iscomparable to that of the 3-HP/4-HB pathway, while the AcC-PrC-Glutamatepathway has a very low pathway specific activity.

The third pathway, the PrC-KS-Glutamate pathway, shown in FIG. 9L isunique because it is the only pathway that utilizes propionyl-CoAcarboxylase without acetyl-CoA carboxylase. The PrC-KS-Glutamate pathwayhas a pathway specific activity that is comparable to that of the3-hydroxypropionate/4-hydroxybutyrate cycle.

The PyrS-ICDH-Glutamate pathway, shown in FIG. 5M, was not furtheranalyzed.

Importantly, all the glutamate cycles utilizes the enzymemethylaspartate mutase. This enzyme is dependent on vitamin B12(cobalamin) for its operation. Eukaryotic photosynthetic organisms donot synthesize this co-factor. Higher plants make use ofcobalamin-independent enzymes and algae, like animals, have arequirement for an external source of vitamin B12. Therefore, theutilization of the glutamate cycles should be restricted tocyanobacteria only.

The Threonine Cycles

FIG. 5I displays carbon fixation pathways which utilize threoninealdolase. Alternatively, threonine can be converted to2-amino-3-oxobutanoate, which is then metabolized to acetyl-CoA andglycine. Using an amino-transferase enzyme, glycine is converted toglyoxylate. The starting metabolite, pyruvate, can be produced either byusing the enzyme pyruvate synthase or by the alanine bypass (see FIG.8). Importantly, the threonine pathways can be considered as a bypass ofthe cycle shown in FIG. 3, the rTCA “shortcut”.

Algorithm for Finding Carbon Fixation Cycles

Stoichiometric properties of biochemical reactions are consideredstructural invariants, unlike the kinetic parameters which are affectedby enzyme concentrations, activity and many other factors. There is avast amount of reliable data for stoichiometric values of virtually allknown enzymes, for example in the KEGG database(www(dot)keggsotjp/kegg/kegg2(dot)html) (Kanehisa M & Goto S (2000)KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res28(1):27-30).

The present inventors developed an algorithm that is a variant ofconstraint based modeling (Papin J A, et al. (2004) Trends Biotechnol22(8):400-405; Schuster S, Dandekar T, & Fell D A (1999) TrendsBiotechnol 17(2):53-60; Schilling C H, Letscher D, & Palsson B O (2000)J Theor Biol 203(3):229-248). The goal of the present algorithm was tofind carbon fixation cycles, i.e. a set of reactions that togethersatisfy the stoichiometric requirements of carbon fixation, which aretransforming three inorganic carbon molecules to one GASP molecule. Theuse of ubiquitous compounds and co-factors (such as H₂O, ATP, NAD(P))was intentionally not taken into consideration.

The first stage was to construct a universal stoichiometric matrix,using the standard representation [Heinrich R & Schuster S (1996) TheRegulation of Cellular Systems (Springer)] which is described below. Thedata was solely taken from KEGG, and stored in the 5280 by 15848 matrix,denoted by S. Note that every reaction is considered to be reversible,and thus is represented by two columns; one for each direction.

In order to reduce the complexity of the algorithm and the amount oftime needed, compounds were removed according to two criteria. First,all the known co-factors were discarded. These are listed in the tablegiven below. Although co-factors play a big role in the energetic costof a cycle and its thermodynamic feasibility, the present inventorswanted at this stage only to find cycles that are stoichiometricallybalanced and leave the other factors for later analysis. The rows in Scorresponding to these co-factors were thus removed. The secondcriterion was to discard all compounds which contain more than 10 carbonatoms or that have a non-specific chemical formula (like the compound“amino acid” with the formula C₂H₄NO₂R). The rows corresponding withthese compounds were removed, and so were the columns of reactions thathave such a compound as a substrate or product. The size restriction isnot necessary and results usually do not change even if the thresholdfor the size of compounds is different. Also, small compounds which areattached to carrier molecules (like CoA or THF) were not excluded, e.g.the “acetyl” in acetyl-CoA has only 2 carbon atoms, and thus was notremoved. In addition, if a reaction was not chemically balanced it wasdiscarded as well.

At this stage, S contained ˜1400 rows and ˜3000 columns. The presentinventors added three special columns, which were not stoichiometricallybalanced: input of CO₂, input of HCO₃ ⁻ and output of GA3P. The inputreactions had the value of (+1) at the row corresponding to CO₂ or HCO₃⁻ and zeros in all other places. The last column in S—the outputreaction—had a (−1) at the row corresponding to GA3P and zeroseverywhere else.

The present inventors then searched for solutions to the followinglinear problem:Sv=0v _(i)≥0v _(output)=1minimize Σv _(i)where v is the flux vector, so that v_(i) is the flux going through eachreaction, and v_(output) is the flux of GA3P output (the valuecorresponding to the last column in S). Note that any solution thatfulfills the first 3 requirements is a carbon fixation cycle (as definedby us). Minimizing Σv_(i) is equivalent to minimizing the total flux.

A script for parsing the data from KEGG, analyzing the compounds,discarding the co-factors and large molecules, checking chemical balanceand producing the final S matrix was written in-house using the Pythonprogramming language. To solve the linear problem, the GLPK (GNU LinearProgramming Kit, www(dot)gnu(dot)org/software/glpk/) was used. Theparameters of the problem were fed to the GLPK solver with the help of awrapper module called ctypes-glpk(www(dot)code(dot)google(dot)com/p/ctypes-glpk/).

The GLPK solver normally returns a sparse minimal solution, which meansthat v has only a few non-zero values. In other words, the solution iscomprised of a small set of reactions which have a positive flux(usually integer values). Sparse solutions are useful because they aresimple and implementing them in-vitro or in-vivo is probably easier.

The carboxylation step is the most important step of a carbon fixationcycle (see Example 1). This step is commonly sensitive to the levels ofCO₂ and O₂ which are diffusible gaseous compounds whose concentrationsare hard to regulate. Therefore, the present inventors were interestedin finding cycles that employ a specific set of carboxylating enzymes,the ones which are kinetically superior. In order to achieve this, thepresent inventors simply “shut out” all the other carboxylating enzymes,by forcing the flux vector to have a value of 0 for the reactionscorresponding to these enzymes. Since fixing inorganic carbon is theonly way to get carbon atoms into the cycle, and since present inventorsconstrained it to export GA3P, the solution to the linear problem willhave to use at least one of the carboxylators in the chosen set.

If that set contains a single enzyme, the flux through it will have tobe exactly 3 (due to the conservation rules of carbon atoms).

TABLE 3 List of co-factors which were removed from the stoichiometricmatrix: Arsenate dIMP FADH2 H2O Arsenite Deamino-NAD+ FAD OxygenManganese HO− Ferricytochrome Orthophosphate Cobalt HSO3−Ferrocytochrome Pyrophosphate Selenate Hydrochloric Ferricytochrome NH3Nickel Nitrite Ferrocytochrome CoA Sulfur Sulfate Reduced Tetrahydro-folate Hydrazine Sulfite Oxidized H+ Trimetaphosphate Sulfur Reduced ATPPhosphoramidate Thiosulfate Oxidized ADP Nitrogen Hydrogen Acceptor AMPSelenophosphate Nitric Donor GTP Nitrate Chloride H2O2 GDP HydrofluoricFluoride dATP GMP Tetrathionate Hydroxylamine dADP CTP HI Selenite dAMPCDP Nitrous Hydrogen dGTP CMP Hg Iodine dGDP UTP Bromide Fe3+ dGMP UDPHydrobromic Fe2+ dUTP UMP Chlorate Iron dUDP ITP Chlorite Magnesium dUMPIDP Diimine Mercury(2+) dTTP IMP Halide O2.− dTDP NADPH ThioredoxinSelenium dTMP NADP+ Thioredoxin Selenide dITP NADH Iodide dIDP NAD+

Pathway Analysis Methods—Thermodynamics I. Thermodynamics of CarbonFixation Cycles

1. The Basic Net Reaction and its Energetics

The reductive pentose phosphate (rPP) pathway can be simplified, or“compressed”, to the following reaction, assuming thatglyceraldehyde-3-phosphate (GA3P) is its product:3.CO₂+6.NADPH+5.H₂O+9.ATP⁻³→GA3P⁻+6.NADP⁺+2.H⁺+9.ADP⁻²+8.P_(i) ²⁻

To better understand the overall reaction, we can be divided it intothree reactions:3.CO₂+12.ē+P_(i) ⁻²+13.H³⁰→GA3P⁻+4.H₂O Inorganic carbon reduction6.NADPH→6.NADP⁺+6.H⁺+12·ē NADPH oxidation9.ATP⁻³+9.H₂O→9.ADP⁻²+9.P_(i) ⁻²+9.H⁺ATP hydrolysis

We are interested in the thermodynamics of carbon fixation under aspecified pH (fixed H⁺ concentration); in other words, we look for thestandard transformed Gibbs energy change (ΔG′_(r)). Under theseconditions the above equation should be changed as follows: (A) Hydrogenatoms are not conserved in the reaction and therefore should not beincluded. (B) Each reactant that can exist in various protonated speciesis represented by a single compound. For example, ‘ATP’ correspond thefollowing protonated species: ATP⁴⁻, HATP³⁻, H₂ATP²⁻. (C) The reactantsdo not show ionic charges or number of hydrogen atoms they containbecause under a constant pH each reactant exists in a superposition ofthose states.

Therefore, under specified and fixed pH, the above reaction should bewritten as:3.CO₂ ^(aq-tot)+6.NADP_(red)+9.ATP+2.H₂O→GA3P+6.NADP_(oxd)+9.ADP+8.P_(i)

The reactant CO₂ ^(aq-tot) refers to a superposition of CO₃ ²⁻(aq), HCO₃⁻(aq), CO₂(aq) and H₂CO₃(aq), where the ratio between those states isdetermined by the system's pH (as well as temperature and ionicstrength).

For convenience in later analysis, the above reaction can be separatedinto two components: an ATP free reaction and an ATP hydrolysisreaction. We shall refer to the ATP-free reaction as the basic carbonfixation net reaction. The basic carbon fixation net reaction and theATP hydrolysis reaction are given by:3.CO₂ ^(aq-tot)+6.NADP_(red)+P_(i)→GA3P+6.NADP_(oxd)+7.H₂O9.ATP+9.H₂O→9.ADP+9.P_(i)

Moreover, assuming that GA3P is the common product of all the carbonfixation pathways analyzed, all can be simplified to the basic netreaction coupled to ATP hydrolysis reactions:X.ATP+X.H₂O→X.ADP+X·P_(i) where X depends on the particular pathway.In order to calculate the standard transformed energies (ΔG_(r)′⁰) ofthe basic net reaction and the ATP hydrolysis reaction one need toobtain the standard transformed energies of formation (ΔG_(r)′⁰) of thereactants participating in those reactions. Those, in turn, can becalculated, at specified pH and ionic strength, from the standardenergies of formation) (ΔG_(f) ⁰), as given by eq. 1:ΔG′ _(f) ⁰ =ΔG _(f) ⁰(I=0)+N _(H) RT ln(10)·pH−(2.91482(z ² −N _(H))I^(0.5))/(1+1.6I ^(0.5))where I, N_(H) and z refer to ionic strength, number of hydrogen atomsin the compound and charge of the compound, respectively. Note that thesecond term actually equals N_(H)(RT ln(10)·pH+ΔG_(f) ⁰(H⁺)), but ΔG_(f)⁰(H⁺) is taken to be 0 in standard calculation and measurements.ΔG_(f)′⁰ is a monotonically increasing function with pH and amonotonically decreasing function with I. The pH affects ΔG_(f)′⁰ moresignificantly than the ionic strength; the second term in the equationis considerably higher than the third one under common physiologicalconditions (5<pH<9, 0<I<0.4, 0<N_(H)<30, |z|≤4). When a certain compoundcan exists in several states, each having a different number of hydrogenatoms (such as CH₃COOH and CH₃COO⁻), the standard transformed energy offormation of this compound is given by eq. 2:

$\mspace{79mu}{{\Delta\; G_{f}^{\prime 0}} = {{- {RT}}\;{\ln\left( {\sum\limits_{j = 1}^{m}\;{\exp\left( {{- \Delta}\;{G_{f}^{\prime 0}(j)}\text{/}{RT}} \right)}} \right)}}}$${\Delta\; G_{f}^{\prime 0}} = {{\Delta\;{G_{f}^{0}\left( {I = 0} \right)}} - {{RT}\;{\ln\left( {\sum\limits_{j = 1}^{m}\;{\exp{\quad\left( {{N_{H}{RT}\;{{\ln(10)} \cdot {pH}}} - \left. \quad{\left( {2.91482\left( {z^{2} - N_{H}} \right)I^{0.5}} \right)\text{/}\left( {1 + {1.6I^{0.5}}} \right)\text{/}{RT}} \right)} \right)}}} \right.}}}$where ΔG_(f) ⁰(j), j=1, 2 . . . m, are the standard transformed energiesof formation of the different states the compound can exist in.

Using this method we have calculated ΔG_(r)′⁰ of the basic net reactionand the ATP hydrolysis reaction in pH ranging from κ to 9 and ionicstrength ranging from 0 to 0.4M. For example:

TABLE 4 ATP hydrolysis Basic net reaction Δ_(r)G′° (KJ/mol), Δ_(r)G′°(KJ/mol), 298.15K, 100 kPa 298.15K, 100 kPa pH = 8 pH = 7 pH = 6 pH = 8pH = 7 pH = 6 −42 −37 −34 196 146 104 I = 0.1M −41 −36 −33 207 157 113 I= 0.25M

ΔG_(r)′⁰ of the basic net reaction is a monotonically increasingfunction with pH and ionic strength. Why is it? Assuming that allreactants and products exist in only one state (eq. 1), the behavior ofΔG_(r)′⁰ as a function of pH depends on the difference between the sumof N_(H) of the reactants and the sum of N_(H) of the products. In thesame manner, the behavior of ΔG_(r)′⁰ as a function of ionic strengthdepends on the difference between the sum of (z²−N_(H)) of the reactantsand the sum of (z²−N_(H)) of the products. If some of the reactants andproducts can exists in several states (eq. 2), no closed solution can beprovided as above. However, in such cases an estimation can be proposed.Usually one of the possible states of each reactants/products is thedominant one (having lower ΔG_(f)′⁰(j)); we can therefore approximateΔG_(f)′⁰ of this compound to that lower ΔG_(f)′⁰(j), as given in eq. 1.

In the case of the basic net reaction ΣN_(H) of the reactants is lowerthan that of the products, while Σ(z²−N_(H)) of the reactants is higherthan that of the products. Therefore, ΔG_(r)′⁰ of the basic net reactionis a monotonically increasing function with pH and ionic strength. Forthe ATP hydrolysis reaction these trends are reversed; ΔG_(r)′⁰ is amonotonically decreasing function with pH because ΣN_(H) of thereactants is higher than that of the products. Also, ΔG_(r)′⁰ is amonotonically decreasing function with ionic strength becauseΣ(z²−N_(H)) of the reactants is lower than that of the products.

In order to calculate the transformed energies ΔG′_(r) (non-standardconcentrations) we need estimations of the reactant concentrations.Following measurements from Spinach chloroplast (42, 43) we have used[GA3P]=0.025 mM, [NADP_(red)]=0.29 mM, [NADP_(oxd)]=0.59 mM, [ATP]=1.9mM, [ADP]=0.76 mM and [P_(i)]=20 mM. The concentration of inorganiccarbon, [CO₂ ^(aq-tot)], was calculated using the apparent Henry's lawconstant, derived from ΔG_(r)′⁰ of the reaction CO₂ ^(aq-tot)⇄CO₂^(g)+H₂O (41). CO₂ ^(g) concentration was assumed to be 387 ppm. FIG.10A displays the expected concentration of total dissolved inorganiccarbon as a function of pH and ionic strength. pH has an extremelystrong effect on this concentration. The [CO₂ ^(aq-tot)] values obtainedby this analysis agree with known experimental data, which measuredbicarbonate concentration of 0.26 mM and 2 mM at pH of 7.4 and 8.2,respectively.

Using these values we have calculated ΔG_(r)′⁰ at the same pH and ionicstrength ranges. For example:

TABLE 5 ATP hydrolysis Basic net reaction Δ_(r)G′ (KJ/mol), Δ_(r)G′(KJ/mol), 298.15K, 100 kPa 298.15K, 100 kPa pH = 8 pH = 7 pH = 6 pH = 8pH = 7 pH = 6 −46 −41 −39 243 209 178 I = 0.1M −46 −41 −38 252 219 187 I= 0.25M

At a given pH and ionic strength one can therefore determine the minimumnumber of ATP hydrolysis reactions needed to be coupled to carbonfixation in order to ensure its feasibility. FIG. 2A presents thisminimal ATP requirement, as a function of pH and ionic strength.Clearly, carbon fixation pathways that can be simplified to the basicnet reaction need to be coupled to the hydrolysis of at least 5-6 ATPmolecules in order to become thermodynamically feasible, under ambientCO₂ ^(g) concentration and characteristic pH and ionic strength. Theextra ATP molecules hydrolyzed by the rPP cycle are not essential forthe thermodynamic feasibility of the cycle but they create an extraenergetic driving force that facilitates carbon fixation whileintroducing several irreversible reactions (bisphosphatases).

2. Modifications of the Basic Net Reaction

Many of the carbon fixation pathways can be simplified to a modifiedform of the basic net reaction, where different redox carriers, otherthan NAD(P)_(red), are utilized by the pathways. Because diverseelectron donors are characterized by different reduction potential,those modifications can significantly change the overall thermodynamicsof the pathways.

2.1 Ferredoxin

Ferredoxin (Fd) is a carrier of only one electron and therefore twoferredoxins are needed in order to replace one NADP_(red) molecule.2.Fd_(red) can replace NADP_(red) corresponding to the use of theenzymes pyruvate and 2-ketoglutarate synthases. ΔG_(r)′⁰ for themodified net reaction can be deduced by treating it as two reactions.The first reaction includes the reduction of NADP_(oxd) by 2.Fd_(red)and the second corresponds to the basic carbon fixation net reaction. Weexemplify this by a modified net reaction in which one NADP_(red) wasreplaced with 2.Fd_(red):2.Fd_(red)+NADP_(oxd)→2.Fd_(oxd)+NADP_(red)3.CO₂ ^(aq-tot)6.NADP_(red)+P_(i)→GA3P+6.NADP_(oxd)+7.H₂OTotal:3.CO₂^(aq-tot)+5.NADP_(red)+2.Fd_(red)+P_(i)→GA3P+5.NADP_(oxd)+2.FD_(oxd)+7.H₂OThe ΔG_(r)′⁰ of the ferredoxin-free reaction was discussed above, andthe ΔG_(r)′⁰ of the first reaction, which reduced NADP_(oxd) by2.Fd_(red), can be calculated as explained above. For example:

TABLE 6 2 · Fd_(red) + NADP_(oxd) → 2 · Fd_(oxd) + NADP_(red) Δ_(r)G′°(KJ/mol), 298.15 K, 100 kPa pH = 8 pH = 7 pH = 6 −9 −15 −21 I = 0.1M −11−17 −22 I = 0.25M

In order to calculate ΔG_(r)′ of the ferredoxin pathways, we need anestimation of the relative concentration of Fd^(red) and Fe^(oxd).Unfortunately, such data is not available. Therefore, we have assumed[Fe^(red)]˜[Fd^(oxd)], which gave, for example:

TABLE 7 2 · Fd_(red) + NADP_(oxd) → 2 · Fd_(oxd) + NADP_(red) Δ_(r)G′(KJ/mol), 298.15 K, 100 kPa pH = 8 pH = 7 pH = 6 −11 −17 −23 I = 0.1M−13 −18 −24 I = 0.25M

This result indicates that the energetic gain of using Fd instead ofNADP, as electron donor, is roughly equivalent to the energy released inthe hydrolysis of one half of an ATP molecule.

2.2 Ubiquinone and Succinate Dehydrogenase

The enzyme succinate dehydrogenase uses ubiquinone (UQ) as an electronacceptor, instead of NADP. Therefore, the net reactions of pathways thatutilize this enzyme include UQ. Those reactions can also be divided intotwo coupled reactions. One includes the reduction of UQ_(oxd) byNADP_(red) and the other corresponds to the basic carbon fixation netreaction. For example:UQ_(oxd)+NADP_(red)→UQ_(red)+NADP_(oxd)3.CO₂ ^(aq-tot)+6.NADP_(red)+P_(i)→GA3P+6.NADP_(oxd)+7.H₂OTotal:3.CO₂^(aq-tot)+7.NADP_(red)+UQ_(oxd)+P_(i)→GA3P+7.NADP_(oxd)+UQ_(red)+7.H₂OThe ΔG_(r)′⁰ of the first reaction, which reduced UQ_(oxd) byNADP_(red), can be again calculated as above. For example:

TABLE 8 UQ_(oxd) + NADP_(red) → UQ_(red) + NADP_(oxd) Δ_(r)G′° (KJ/mol),298.15 K, 100 kPa pH = 8 pH = 7 pH = 6 −65 −71 −77 I = 0.1M −64 −69 −75I = 0.25MDue to a lack of available data we again assume [UQ_(oxd)]˜[UQ_(oxd)].For example:

TABLE 9 UQ_(oxd) + NADP_(red) → UQ_(red) + NADP_(oxd) Δ_(r)G′ (KJ/mol),298.15 K, 100 kPa pH = 8 pH = 7 pH = 6 −64 −69 −75 I = 0.1M −62 −68 −73I = 0.25M2.3 Menaquinone and Fumarate Reductase

The enzyme fumarate reductase can use several redox curriers as electrondonors. Those include menaquinone (MQ) FAD/FMN or even NAD_(red).Menaquinone is the common electron donor for membrane-bound fumaratereductase. Applying the same reaction division procedure as above weget:MQ_(red)+NADP_(oxd)→MQ_(oxd)+NADP_(red)3.CO₂ ^(aq-tot)+6.NADP_(red)+P_(i)→GA3P+6.NADP_(oxd)+7.H₂OTotal:3.CO₂^(aq-tot)+5.NADP_(red)+MQ_(red)P_(i)→GA3P+5.NADP_(oxd)+MQ_(oxd)+7.H₂OWe weren't able to find information on ΔG_(f)′⁰ (MQ_(red/oxd)) asfunction of pH and ionic strength. Therefore, in order to calculate theenergy change Δ_(r)G′⁰ of the first reaction, we have used the reductionpotential of menaquinone, −75 mV (47, 48):ΔE(MQ_(red)+NADP_(oxd)→UQ_(oxd)+NADP_(red))=−245 mVHence we obtain: ΔG_(r)′⁰(MQ_(red)+NADP_(oxd)→UQ_(oxd)+NADP_(red))=−2·96.5·−0.245=+47 KJ/mol

This result indicates that the energetic cost of using MQ instead ofNADP, as electron donor, is roughly equivalent to the energy released inthe hydrolysis of one ATP molecule. Eukaryotic organisms usually userhodoquinone instead of menaquinone. The reduction potential ofrhodoquinone is −63 mV, very close to that of menaquinone.

Intuitively, one might think that because MQ is ultimately reduced usingNADP_(red) we can neglect MQ altogether and refer to NADP_(red) as theelectron donor de-facto. However, in most organisms that employ fumaratereductase MQ is being reduced by the oxidation of NADP_(red) by therespiratory complex I. The electron flow through part of complex I isused to generate proton gradient and hence ATP. This energetic couplingdisrupts the thermodynamic calculation by increasing the overallΔG_(f)′⁰ and making the energetics of MQ reduction by NADP_(red) verydifficult to calculate. Therefore, we regard MQ_(red), and notNADP_(red), as the electron donor.

The energetic cost of using menaquinone can be avoided by employingfumarate reductase enzymes that utilize other electron donors. Fumaratereductase from Saccharomyces cerevisiae is a soluble enzyme that acceptselectrons from a non-bound, reduced FAD. The energetic cost of using FADcan be calculated as above. For example:

TABLE 10 FAD_(red) + NADP_(oxd) → FAD_(oxd) + NADP_(red) Δ_(r)G′°(KJ/mol), 298.15 K, 100 kPa pH = 8 pH = 7 pH = 6 14 20 26 I = 0.1M 13 1824 I = 0.25M

Due to a lack of available data we again assume [FAD_(oxd)]˜[FAD_(oxd)],arriving at:

TABLE 11 FAD_(red) + NADP_(oxd) → FAD_(oxd) + NADP_(red) Δ_(r)G′(KJ/mol), 298.15 K, 100 kPa pH = 8 pH = 7 pH = 6 13 18 24 I = 0.1M 11 1722 I = 0.25M

Several organisms are known to use NAD_(red) as a direct electron donorfor fumarate reductase, avoiding any additional energetic cost.Importantly, this enzyme variant is soluble and remains active underaerobic conditions. We would expect organisms that reduce fumarate aspart of the reductive TCA to employ this efficient enzyme. Indeed, arecent paper has demonstrated that this is the case for the thermophilicbacterium, Hydrogenobacter thermophilus, which fixes carbon through thereductive TCA cycle.

3. Energetic of Carbon Fixation Pathways

Employing the modifications analyzed above we were able to calculateΔG′_(r) for the carbon fixation pathways, both natural and synthetic.Almost all pathways are thermodynamically feasible at ambient CO₂ ^(g)concentration (Δ_(r)G^(o)<<0), as shown in Table 1, FIG. 12. However,the natural rTCA cycle and rAcCoA pathway (shown in FIG. 5B) are notfeasible (Δ_(r)G^(o)>0) at a broad range of pH and ionic strengths andunder ambient CO₂ ^(g) concentration as shown in FIG. 10B.

Notably, increased CO₂ ^(g) concentration can make those pathwaysfeasible; the rTCA-MQ cycle becomes feasible at all pH and ionicstrength values at X100 the CO₂ ^(g) ambient concentration, while therAcCoA pathway is feasible at pH<8 at the same CO₂ ^(g) concentration(FIG. 10B). Such elevated CO₂ ^(g) concentration might be expected incertain environments, in C4 and CAM plants and in algae that use CO₂concentrating mechanisms.

Importantly, three of the enzymes of the rAcCoA pathway can usemolecular hydrogen as direct electron donors, instead of NAD(P)H. We didnot calculate the energetics of such carbon fixation process due to thewide range of H₂ concentrations that can be present in the medium,spanning many magnitudes of orders.

It should be noted that the rTCA cycle and the rAcCOA pathway operate instrictly anaerobic organisms. Anaerobes are energy restricted ascompared to aerobes because they utilize only chemical reactions withlow energetic yield (e.g. sulfur, Fe, Mn and nitrate respirations,).This limits the availability of energy that they can invest in carbonfixation. In order to achieve thermodynamic feasibility and sustaingrowth, the organisms that utilize either of those cycles must occupyhigh CO₂ habitats or operate a carbon concentrating mechanism. Moreover,the reductive acetyl-CoA pathway, is known to be coupled to otherexogenic cellular processes in order to achieve energetic feasibility.

4. Carbon Fixation Cycles can be “Distributed ThermodynamicBottlenecks’”

The analysis of energetics described above refers to the whole carbonfixation pathway as a single unit. This can predict the infeasibility ofa pathway. It can also predict its feasibility in cases where we do notrestrict the concentrations of the different metabolites. In practicehowever, those concentrations are constrained by both upper and lowerlimits. As a result, the overall energetics of the pathway is not enoughto predict feasibility. A sub-pathway within the pathway can beinfeasible under given set of concentration restrictions even if theΔG_(r) of the entire pathway is less than 0. The sub-pathway is thenreferred to as a “distributed thermodynamic bottleneck”.

We would like to check whether the cycle of each pathway creates adistributed thermodynamic bottleneck. There are four types of cycles,classified by their products; their basic net reactions are given below(not including ATP hydrolysis). The energy changes ΔG_(r)′⁰ of thesereactions were calculated as explained above. For example:

TABLE 12 Standard Transformed Gibbs Energy Changed for Carbon FixationCycles Δ_(r)G′° (KJ/mol), 298.15 K, 100 kPa Reaction/ pH = 8 pH = 7 pH =6 Modification Glyoxylate-forming cycles: 2 · CO₂ ^(aq-tot) + 2 ·NADP_(red) → Glyoxylate(C₂O₃) + 2 · NADP_(oxd) + 3 · H₂O 90 73 60 I =0.1M 93 76 63 I = 0.25M Acetyl-CoA-forming cycles: 2 · CO₂ ^(aq-tot) + 4· NADP_(red) + CoA → Acetyl-CoA(C₂O-CoA) + 4 · NADP_(oxd) + 5 · H₂O 38−2 −38 I = 0.1M 45 5 −31 I = 0.25M Pyruvate-forming cycles: 3 · CO₂^(aq-tot) + 5 · NADP_(red) → Pyruvate(C₃O₃) + 5 · NADP_(oxd) + 6 · H₂O82 43 8 I = 0.1M 90 51 16 I = 0.25M Oxalate-forming cycles: 2 · CO₂ +NADP_(red) → Oxalate(C₂O₄) + NADP_(oxd) + 2 · H₂O 49 44 42 I = 0.1M 5045 42 I = 0.25M

The concentrations of the cycles' products, as well as that of CoA, wereestimated to lie between 1 μM and 10 mM. For CoA, acetyl-CoA andpyruvate, those estimations are supported by experimental data. Forglyoxylate and oxalate, those estimations correspond to the affinitiesof those metabolites to their utilizing enzymes; the affinity ofglyoxylate towards glyoxylate carboligase is 250 μM, while the affinityof oxalate towards the enzyme oxalate-coA ligase is 2 mM. Themaximal/minimal ratio between CoA and acetyl-CoA was taken as 10^(±2).

We have calculated ΔG′_(r) for the various cycles, employed by thedifferent pathways, under this broad range of product concentrations.The cycles of most pathways are feasible at all pH and ionic strengthvalues and under all possible product concentration, as shown inTable 1. Notably, the KGS-ICDH and PyrS-ME cycles are not feasible atall pH, ionic strength and product concentration values. Other threeferredoxin-oxidoreductase pathways, KGS-KGC, PyrS-PyrC-Glyoxylate andPrC-KGS-Glutamate, are all non-feasible at some pH and ionic strengthvalues, which are dependent on the estimated glyoxylate concentration(FIG. 10C, upper row). The non-ferredoxin-oxidoreductase-containingpathway MCC-ICDH-Citrate also displays a similar behavior (FIG. 10C,bottom row). However, replacing the CoA-transferase enzyme, whichconverts 2-hydroxyglutaryl-CoA to 2-hydroxyglutarate, by a yet unknownthioester hydrolyze enzyme, will effectively reduce the ΔG of this cyclewell below 0. While at some pH and ionic strength values the acetyl-CoAproducing cycle of the rTCA cycle become infeasible, this constrain isless strict than that imposed on this pathway as a whole (GASP-forming).

D. Pathway Analysis Methods—Kinetics

I. Rate Analysis of Metabolic Pathways

1. The Pathway Specific Activity is the Upper Limit of the Specific Fluxof a Pathway

The specific flux of a pathway is the overall flux sustained by thepathway, J, divided by the total concentration of the enzymes utilizedby the pathway, ΣE_(i)=E_(t). In the general case of non-linearpathways, the flux through individual enzymes is not necessarily thesame. In such case, we assign a stoichiometric coefficient, w_(i), toeach reaction i, which corresponds to the number of catalytic cycles thereaction takes to produce one molecule of the pathway's product (forexample, given the simple pathway E₁:X→Y, E₂:Y+Y→Z, E₃:Z→Product, weassign w₁=2 and w₂=w₃=1.). The enzyme cost (1/V_(i), see methods) foreach reaction is thus multiplied by its stoichiometric coefficient. Tomaintain an overall flux of 1 μmol/min the enzyme cost of the wholepathway is therefore given by

$\Sigma{\frac{w_{i}}{V_{i}}.}$As a result, in the general case, the pathway specific activity is givenby:

${PSA}_{A} = {1\text{/}{\sum\limits_{i = 1}^{m}\;\frac{w_{i}}{V_{i}}}}$where m, V_(i) and w_(i) are the number of the enzymes the pathwayutilizes, the specific activities of those enzymes and theirstoichiometric coefficients in the pathway, respectively. The specificflux of a pathway and the pathway specific activity are both given inunits of mol/min/mg.

The specific flux of a pathway is approximated by the pathway specificactivity if the following three assumptions hold:

-   (I) The backward reactions are negligible as compared to the forward    one.-   (II) All enzymes work in their zero-order regime, namely, they are    substrate-saturated.-   (III) The relative concentrations of the enzymes are optimal, which    means that there is no surplus of any enzyme.

Intuitively, if we assume a non-negligible backward reaction of acertain enzyme its de-facto activity in the forward direction isdecreased. The same holds for an enzyme that is not substrate-saturated;its activity is expected to be lower. Lastly, a non-optimalconcentration of the enzymes also decreases the specific flux of apathway. Therefore, a deviation from each of those assumptions resultsin a specific flux that is lower than the calculated pathway specificactivity. In other words, the pathway specific activity is an upperlimit estimation for the specific flux of a pathway.

The strength of the pathway specific activity as an approximation of thespecific flux lies in that it does not require complete information ofthe kinetic parameters of the enzymes (the Michaelis constants) nor itnecessitates an estimation of the intermediate metaboliteconcentrations.

Below we show the derivation of the specific flux of a simple,non-branching pathway, using different assumption sets. We demonstratethat indeed the pathway specific activity is higher than the specificfluxes calculated under different assumptions.

1.1 Determining the Specific Activities of Enzymes

Information on enzyme kinetics is dispersed and non standardized. As anestimate to determine the specific activities of the enzymes composingthe different pathways we embarked on a comprehensive literature surveyof >1500 papers. For each enzyme all available and relevant values(ranging 1-40 values per enzyme) were obtained. We have discarded thebottom 50% values, which represent less adapted versions and the top 10%values, which might correspond to experimental errors and unnaturalconditions. We took the average of the remaining values as arepresentative specific activity of each enzyme.

The specific activities of the carboxylating enzymes were calculated asfollows: we obtained, from each paper a specific activity value (undersaturating CO₂/HCO₃ ⁻) and carbon species (CO₂ or HCO₃ ⁻) affinity. Wehave calculated the specific activity under ambient CO₂/HCO₃ ⁻concentrations by applying Michaelis-Menten kinetics with nocooperativity: SA_(ambient)=SA_(saturating)[C]/([C]+K_(m) ^(C)), where[C] is the ambient concentration of the carbon species and K_(m) ^(C) isthe affinity of the enzyme towards that carbon species. [C] was taken tobe 10 μM for CO₂ and was taken as 200 μM for HCO₃ ⁻, corresponding to apH of 7.2-7.4. The same data filtering as for specific activities of thenon-carboxylating enzymes was applied and the average was set as arepresentative specific activity. Conservatively, for RUBISCO we chose ahigher specific activity than the average value, 1.3 μmole/min/mginstead of 1.13, which correspond to the most recent studies and also tothe well-accepted Kcat values of 3-4 sec⁻¹.

The enzyme list, including representative specific activities and ATPand NAD(P)H requirements is provided below in section G. Overall, 109reactions were analyzed and over 1500 papers were scanned.

2. A Simple Pathway: General Definitions

$\begin{matrix}{S_{0}\underset{v_{1}}{\Leftrightarrow}S_{1}\underset{v_{2}}{\Leftrightarrow}S_{2}\underset{v_{3}}{\Leftrightarrow}\ldots\underset{v_{n - 1}}{\Leftrightarrow}S_{n - 1}\underset{v_{n}}{\Leftrightarrow}S_{n}} & (1) \\{{\frac{{dS}_{i}}{dt} = {v_{i} - v_{i + 1}}},{i = 1},\ldots\mspace{14mu},n} & (2)\end{matrix}$

The concentration of the pathway substrate is assumed to be fixed, S0=S.The concentration of the product is assumed to be zero (a strongmetabolic sink), Sn=0.

In all further analysis we assume that the system is in a steady state.In this case J=v_(i) for any i.

The Haldane reversible three-step model (62) states:

$\begin{matrix}{v_{i} = {E_{i}\frac{{V_{i}^{+}\frac{S_{i - 1}}{K_{i}^{+}}} - {V_{i}^{-}\frac{S_{i}}{K_{i}^{-}}}}{1 + \frac{S_{i - 1}}{K_{i}^{+}} + \frac{S_{i}}{K_{i}^{-}}}}} & (3)\end{matrix}$where v_(i) ⁺ and v_(i) ⁻ are the maximal specific forward and backwardrates (μmol/min/mg), respectively, and E_(i) is the amount of enzyme I(mg). K_(i) ⁺ and K_(i) ⁻ are the Michaelis constants for the substrateand the product (mM) and S_(i−1) and S_(i) are the concentrations of thesubstrate and the product (mM), respectively. Throughout the analysis wewill assume a total volume of 1 litter, which imposes the same flux inunits of mol/min/mg or in units of μM/min/mg.

We would like to maximize J/E_(T).

3. Saturating Substrate (Zero-Order) Estimation

As mentioned above, two assumptions lie behind the pathway specificactivity analysis:

-   (I) The backward reaction is negligible as compared to the forward    one, V_(i) ⁺/K_(i) ⁺>>V_(i) ⁻/K_(i) ⁻.-   (II) All enzymes work in their zero-order regime, K_(i) ⁺<<S_(i+1).

Those assumptions yield a steady-state flux ofJ=v _(i) =E _(i) V _(i) ⁺  (4)which in turn imposes an optimal enzyme distribution of

$\begin{matrix}{\frac{E_{1}}{E_{T}} = {\left( {1\text{/}V_{i}^{+}} \right)/{\sum\limits_{j = 1}^{i}\;\left( {1\text{/}V_{j}^{+}} \right)}}} & (5)\end{matrix}$

In such an optimal enzyme distribution no enzyme is in surplus and therelative amount of each enzyme is a function of the rates of all theother enzymes, but of no other parameter.

Substituting E_(i) in J yields

$\begin{matrix}{\frac{J}{E_{T}} = {1/{\sum\limits_{j = 1}^{i}\;\left( {1\text{/}V_{j}^{+}} \right)}}} & (6)\end{matrix}$

Considering n identical reactions, with equipotent rates V⁺=V, wefinally get:

$\begin{matrix}{\frac{J}{E_{T}} = {\frac{1}{n}V}} & (7)\end{matrix}$4. Linear-Regime Estimation

Let us assume S_(i−1)<<K_(i) ⁺, S_(i)<<K_(i) ⁻.

The rate of each reaction can therefore be expressed using linear rateconstants (63, 64):

$v_{i} = {{{k_{i}S_{i - 1}} - {k_{- i}S_{i}}} = {k_{i}\left( {S_{i - 1} - \frac{S_{i}}{q_{i}}} \right)}}$where${k_{i} = {\frac{V_{i}^{+}}{K_{i}^{+}}E_{i}}},{k_{- i} = {\frac{V_{i}^{-}}{K_{i}^{-}}E_{i}}},{q_{i} = \frac{k_{i}}{k_{- i}}}$

In such a case the steady-state flux and the intermediates'concentrations are given by

$\begin{matrix}{J = {\left( {S{\prod\limits_{j = 1}^{n}\; q_{j}}} \right)/\left( {\sum\limits_{j = 1}^{n}\;{\frac{1}{k_{j}}{\prod\limits_{m = j}^{n}\; q_{m}}}} \right)}} & (8) \\{S_{k} = {{S{\prod\limits_{j = 1}^{k}\; q_{j}}} - {J{\sum\limits_{l = 1}^{k}\;{\frac{1}{k_{l}}{\prod\limits_{j = l}^{k}\; q_{j}}}}}}} & (9)\end{matrix}$

Maximizing J/E_(T) gives the following enzyme distribution (63, 64)

$\begin{matrix}{\frac{E_{i}}{E_{T}} = {\left( \sqrt{\frac{1}{V_{i}^{+}}{\prod\limits_{j = 1}^{i - 1}\;\frac{1}{q_{j}}}} \right)/\left( {\sum\limits_{k = 1}^{n}\;\sqrt{\frac{1}{V_{k}^{+}}{\prod\limits_{j = 1}^{k - 1}\;\frac{1}{q_{j}}}}} \right)}} & (10)\end{matrix}$

In order to compare the results obtained from different assumptions weconsider n identical reactions, with equipotent rates and constants:K_(i) ⁺=K⁺, V_(i) ⁺=V⁺, K_(i) ⁻=K⁻, V_(i) ⁻=V⁻.

4.1. Equipotent Forward and Backward Rates, q=1

The optimal enzyme distribution in this case is E_(i)=E_(T)/n.Substituting in eq. 8 gives

$\begin{matrix}{\frac{J}{E_{T}} = {\frac{S}{E_{T}\frac{1}{k}n} = {\frac{S}{E_{T}\frac{K}{V\frac{E_{T}}{n}}n} = {\frac{1}{n^{2}}V\frac{S}{K}}}}} & (11)\end{matrix}$4.2. Forward Reactions Faster than Backward Reactions, q>1

$\begin{matrix}{\frac{J}{E_{T}} = {{\left( {S{\prod\limits_{j = 1}^{n}\; q_{j}}} \right)/\left( {E_{T}{\sum\limits_{j = 1}^{n}\;{\frac{1}{k_{j}}{\prod\limits_{m = j}^{n}\; q_{m}}}}} \right)} = {{{Sq}^{n}\text{/}\left( {E_{T}\frac{K}{V}{\sum\limits_{j = 1}^{n}\;\frac{q^{n - j + 1}}{E_{j}}}} \right)} = {V{\frac{S}{K}/\left( {E_{T}{\sum\limits_{j = 0}^{n - 1}\;\frac{1}{q^{j}E_{j + 1}}}} \right)}}}}} & (12)\end{matrix}$4.2.1. Optimal Enzyme Distribution

$\begin{matrix}{\frac{E_{i}}{E_{T}} = {{\left( \sqrt{\frac{1}{q^{i - 1}}} \right)/\left( {\sum\limits_{k = 1}^{n}\;\sqrt{\frac{1}{q^{k - 1}}}} \right)} = {{\frac{1}{q^{\overset{i}{2}}}/\left( {\sum\limits_{k = 1}^{n}\;\left( \frac{1}{q} \right)^{\overset{k}{2}}} \right)} = {\frac{1}{q^{\overset{i}{2}}}\frac{q^{\overset{n + 1}{2}} - q^{\overset{2}{n}}}{q^{\overset{n}{2}} - 1}}}}} & (13)\end{matrix}$

Substitution of eq. 13 in eq. 12 yields

$\begin{matrix}{\frac{J}{E_{T}} = {{V{\frac{S}{K}/\left( {\sum\limits_{j = 0}^{n - 1}\;{\frac{q^{\overset{j + 1}{2}}}{q^{j}}\frac{q^{\overset{n}{2}} - 1}{q^{\overset{n + 1}{2}} - q^{\overset{n}{2}}}}} \right)}} = {V\frac{S}{K}{E_{t}\left( \frac{q^{\overset{n}{2}} - q^{\overset{n - 1}{2}}}{q^{\overset{n}{2}} - 1} \right)}^{2}}}} & (14)\end{matrix}$which, for q>1, is a monotonically increasing function with q thatobeys:

$\begin{matrix}{{\frac{1}{n^{2}}V\frac{S}{K}} < \frac{J}{E_{t}} < {V\frac{S}{K}}} & (15) \\{{\frac{J}{E_{t}}\overset{q\rightarrow 1}{\rightarrow}{\frac{1}{n^{2}}V\frac{S}{K}}},{\frac{J}{E_{t}}\overset{q\rightarrow\infty}{\rightarrow}{V\frac{S}{K}}}} & (16)\end{matrix}$4.2.2. Non-Optimal, Uniform, Enzyme Distribution E_(i)=1/n

$\begin{matrix}{\frac{J}{E_{T}} = {{V{\frac{S}{K}/\left( {n{\sum\limits_{j = 0}^{n - 1}\;\frac{1}{q^{j}}}} \right)}} = {V\frac{S}{K}\frac{1}{n}\frac{q^{n} - q^{n - 1}}{q^{n} - 1}}}} & (17)\end{matrix}$which, for q>1, is a monotonically increasing function with q thatobeys:

$\begin{matrix}{{\frac{1}{n^{2}}V\frac{S}{K}} < \frac{J}{E_{T}} < {\frac{1}{n}V\frac{S}{‘K’}}} & (18) \\{{\frac{J}{E_{T}}\overset{q\rightarrow 1}{\rightarrow}{\frac{1}{n^{2}}V\frac{S}{K}}},{\frac{J}{E_{T}}\overset{q\rightarrow\infty}{\rightarrow}{\frac{1}{n}V\frac{S}{K}}}} & (19)\end{matrix}$4.2.3 Osmotic Constraint

Still considering a linear-regime and optimizing J/E_(T), we add anotherconstraint, on the overall concentration of the intermediates:ΣS_(i)=Ω⁰.

In such case and when q→∞ the optimal enzyme distribution obeys (63)

$\begin{matrix}{{\frac{E_{1}}{E_{T}} = \frac{\Omega^{0}}{\Omega^{0} + {n^{2}S}}},{\frac{E_{i}}{E_{T}} = {\frac{nS}{\Omega^{0} + {n^{2}S}}\mspace{14mu}\left( {2 \leq i \leq n} \right)}}} & (20)\end{matrix}$

In such case the specific flux is given by

$\begin{matrix}{\frac{J}{E_{T}} = {{V{\frac{S}{K}/\left( {\sum\limits_{j = 0}^{n - 1}\;\frac{1}{q^{j}E_{j + 1}}} \right)}} = {{V{\frac{S}{K}/\left\lbrack {\left( {\Omega^{0} + {n^{2}S}} \right)\left( {\frac{1}{\Omega^{0}} + {\frac{1}{nS}\frac{q^{n - 1} - 1}{q^{n} - q^{n - 1}}}} \right)} \right\rbrack}}\underset{q\rightarrow\infty}{\rightarrow}{V\frac{S}{K}\frac{\Omega^{0}}{\Omega^{0} + {n^{2}S}}}}}} & (21)\end{matrix}$

We shell now assume (practically) Ω⁰˜S

$\begin{matrix}{\frac{J}{E_{T}}\underset{\Omega_{S}^{0}\rightarrow 1}{\rightarrow}{\frac{1}{1 + n^{2}}V\frac{S}{K}}} & (22)\end{matrix}$5. Summary of Analysis

Table 13 shown below compares the specific fluxes obtained usingdifferent assumptions.

The factor S/K (>1) separates the results of the linear-order assumptionfrom the zero-order assumption, which indeed corresponds to theindependence of the second assumption on both substrate concentrationand Michaelis constants.

The pre-factors that emerge from the different assumption sets seem tolie between the asymptotes 1/n² and 1/n. Indeed, the pre-factor of eq.15 and 16 (linear-order, q>1, optimize enzyme distributions) tends to 1with increasing q. However, increasing q, in this case, is coupled withan increased imbalance of the enzymes' distribution and with asuperlinearly increase of the overall intermediate concentrations (63),which very quickly break the liner-order assumption. In-vivo, thoseeffects are unrealistic. Indeed, introducing an osmotic constraintresults in pre-factor of 1/(1+n²), when q→∞. Therefore, the pre-factor1/n, which corresponds to the assumptions we have used in the paper,seems to serve as upper limit estimation.

To conclude, our simplistic analysis indeed indicates that the pathwayspecific activity is an upper limit estimation of the specific flux of apathway.

TABLE 13 Comments Specific Flux Assumptions PSA is a function of Vand n. Michaelis constants and intermediate concentrations are notrelevant. $\frac{J}{E_{T}} = {V\frac{1}{n}}$ Zero-order regime(S_(i−1) >> K_(i) ⁺)${Negligible}\mspace{14mu}{reverse}\mspace{14mu}{reaction}\mspace{14mu}\left( {\frac{V_{i}^{+}}{K_{i}^{+}}\operatorname{>>}\frac{V_{i}^{-}}{K_{i}^{-}}} \right)$${Enzyme}\mspace{14mu}{distribution}\mspace{14mu}{is}\mspace{14mu}{{fixed}{\mspace{11mu}\;}\left( {\frac{E_{1}}{E_{T}} = \frac{\left( \frac{1}{V_{i}^{+}} \right)}{\sum\limits_{j = 1}^{i}\;\left( \frac{1}{V_{j}^{+}} \right)}} \right)}$$\frac{J}{E_{T}} = {\frac{1}{n^{2}}V\frac{S}{K}}$${Linear}\text{-}{order}\mspace{14mu}{regime}\mspace{14mu}\left( {S_{i}^{+ \frac{\bullet}{-}}{\operatorname{<<}K_{i}^{+ \frac{\bullet}{-}}}} \right)$Forward  and  backward  reactions  are equipotent  (q = 1)The  optimal   enzyme  distribution   is${{uniform}{\mspace{11mu}\;}\left( {E_{i} = {\frac{E_{t}\sqrt{\cdots}}{\sum\limits_{k = 1}^{n}\;\sqrt{\cdots}}\; = \frac{1}{n}}} \right)}\mspace{14mu}$The total intermediates concentration increases superlinearly withincreasing q. See text.${\frac{1}{n^{2}}V\frac{S}{K}} < \frac{J}{E_{T}} < {V\frac{S}{K}}$${Linear}\text{-}{order}\mspace{14mu}{regime}\mspace{14mu}\left( {S_{i}^{+ \frac{\square}{-}}{\operatorname{<<}K_{i}^{+ \frac{\square}{-}}}} \right)$Forward reaction is faster than the backward (q > 1)Optimal  enzyme  distribution$\;\left( {E_{i} = \frac{E_{t}\sqrt{\cdots}}{\sum\limits_{k = 1}^{n}\;\sqrt{\cdots}}} \right)$${\frac{1}{n^{2}}V\frac{S}{K}} < \frac{J}{E_{T}} < {\frac{1}{n}V\frac{S}{K}}$${Linear}\text{-}{order}\mspace{14mu}{regime}\mspace{14mu}\left( {S_{i}^{+ \frac{\square}{-}}{\operatorname{<<}K_{i}^{+ \frac{\square}{-}}}} \right)$Forward  reaction  is   faster  than  the backward  (q > 1)${Uniform}\mspace{14mu}{enzyme}\mspace{14mu}{{distribution}\left( {E_{i} = \frac{1}{n}} \right)}$$\frac{J}{E_{T}} = {\frac{1}{1 + n^{2}}V\frac{S}{K}}$${Linear}\text{-}{order}\mspace{14mu}{regime}\mspace{14mu}\left( {S_{i}^{+ \frac{\square}{-}}{\operatorname{<<}K_{i}^{+ \frac{\square}{-}}}} \right)$Forward  reaction  is   faster  than  the backward  (q → ∞)Osmotic  constraint  (ΣS_(i) = Ω⁰, Ω⁰∼S)${Optimal}\mspace{14mu}{enzyme}\mspace{14mu}{{distribution}\left( {{\frac{E_{1}}{E_{T}} = \frac{\Omega^{0}}{\Omega^{0} + {n^{2}S}}},{\frac{E_{1}}{E_{T}} = \frac{nS}{\Omega^{0} + {n^{2}S}}}} \right)}$II. The Stoichiometric Coefficients of the Reductive Pentose PhosphateCycle's Enzymes

In order to determine the stoichiometric coefficients of the enzymes inthe reductive pentose phosphate (rPP) cycle we have assumed a zero netflux of each metabolite (a steady-state assumption). Therefore, we canbuild a set of linear equations that describes the relation between thedifferent enzymatic rates. This set is represented by the followingequation: S_(C×R)·v_(R×i)=b_(C×i), where S is the stochiometry matrix, vis the flow vector (which equals the stoichiometric coefficients), b isa vector corresponding to the change in the concentrations of eachcompound, C is the number of compounds and R is the number of reaction.

As shown in FIG. 5A, the carboxylase reaction flux of RUBISCO is markedby v_(i), while the oxygenase reaction flux is marked by v′₁. Thecarboxygenase reaction of RUBISCO generate two molecules ofglycerate-3-phosphate (G3P) from a single molecule ofribulose-1,5,-bisphosphate (RuBP). The oxygenase reaction, however,produces 1.5 moles of G3P for every mole of RuBP. One mole is formeddirectly from the oxygenase reaction and another half is produced by thephotorespiration pathway that condenses two glycolate-2-phosphatemolecules, which were formed by the oxygenase reaction.

Therefore, the stochiometry matrix, S, is given as follows:

TABLE 14 Reaction # 13 12 11 10 9 8 7 6 5 4 3 2 1′ 1 0 0 0 0 0 0 0 0 0 00 −1 1.5 2 G3P Compound 0 0 0 0 0 0 0 0 0 0 −1 1 0 0 GBP 0 0 0 −1 0 0 −10 −1 −1 1 0 0 0 GA3P 0 0 0 0 0 −1 0 0 −1 1 0 0 0 0 DHAP 0 0 0 0 0 0 0 −11 0 0 0 0 0 FBP 0 0 0 0 0 0 −1 1 0 0 0 0 0 0 F6P 0 0 0 0 0 −1 1 0 0 0 00 0 0 E4P 0 0 0 0 −1 1 0 0 0 0 0 0 0 0 SBP 0 0 0 −1 1 0 0 0 0 0 0 0 0 0S7P 0 −1 0 1 0 0 1 0 0 0 0 0 0 0 X5P 0 0 −1 1 0 0 0 0 0 0 0 0 0 0 R5P −11 1 0 0 0 0 0 0 0 0 0 0 0 Ru5P 1 0 0 0 0 0 0 0 0 0 0 0 −1 −1 RuBP

Glyceraldehyde-3-phosphate (GA3P) is the product of the rPP cycle andtherefore the outward flux v_(out) is a flux of GA3P leaving the cycle(FIG. 5A).

The vector of the change in the concentrations of each compound, b, ishence taken to be:

TABLE 15 0 G3P Compound 0 GBP 1 GA3P 0 DHAP 0 FBP 0 F6P 0 E4P 0 SBP 0S7P 0 X5P 0 R5P 0 Ru5P 0 RuBP

To find the stoichiometric coefficient d, corresponding to the flowvector, v, we need to solve S_(C×R)·v_(R×i)=b_(C×i).

Let us assume a ratio between RUBISCO oxygenase (v′₁) and carboxygenase(v₁) reactions of c.

Solving the equation we get the flux vector v as shown below, whereλ=(1+c)/(1−0.5c). We chose two representing c values. The first is c=0,corresponding to no photorespiration (C4 and CAM plants, elevated CO₂concentration, CO₂ concentrating mechanisms ext.). Secondly, we took thebiologically relevant c=0.25, corresponding to terrestrial C3 plants.The resulting vector v matches the stoichiometric coefficients of therPP cycle's enzymes.

TABLE 16 Reaction # 13 12 11 10 9 8 7 6 5 4 3 2 1′ 1 3λ 2λ λ λ λ λ λ λ λ2λ 5λ + 1 5λ + 1 (λ + 2)c λ + 2 3 2 1 1 1 1 1 1 1 2 6 6 0 3 c = 0 4.292.86 1.43 1.43 1.43 1.43 1.43 1.43 1.43 2.86 8.15 8.15 0.86 3.43 c =0.25III. Correlation Between Pathway Specific Activity and Number of Enzymes

The simplicity of a pathway, which corresponds to the number of enzymesit utilizes, is often taken as an indicator for its specific flux. Thenumber of enzymes is expected to be correlated to the totalconcentration of the pathway's enzymes; therefore simplicity shouldcorrelate with the pathway specific flux.

To test this assumption we analyzed the correlation between the pathwayspecific activities and the (total) number of enzymes of thenon-ferredoxin-oxidoreductase-containing pathways, as given in Table 1,FIG. 14 and shown in FIG. 11. We did not include theferredoxin-oxidoreductase pathways because they have artificially higherspecific activities stemming from our lack of knowledge regarding thekinetic properties of the ferredoxin-oxidoredcutase enzymes.

The linear-correlation between the two criteria gave R²˜0.18 with agradient of ˜0.015. Using a Z-test we get z=R²·(n−3)^(0.5)=0.59, whichgives p-value of 0.28. Therefore, no significant correlation could beestablished between those criteria and hence the simplicity of a pathwaycannot serve as a reliable indicator for its pathway specific activity.

E. Pathway Analysis Methods—Metabolic Compatibility

I. Functional Analysis of the Effects of Novel Cycles on the MetabolicNetwork

1. The Model

In designing alternative CO₂ assimilation pathways, it is important topredict how such pathways will integrate into the rest of the metabolicnetwork and how they influence closely connected metabolic pathways.Constraint-based modeling provides a reliable means of doing such ananalysis. In constraint-based modeling of metabolic networks, successivelayers of known constraints can be outlined to find a solution space ofallowable phenotypes. Such constraints include all known chemicalreactions for an organism, metabolic reaction stoichiometry, allowedreaction directionality, and known uptake and secretion rates. Whilethis modeling framework does not incorporate information about kinetics,it can provide accurate measures of growth yield, secretion products,the viability of environmental and genetic perturbations and manyadditional insights.

To investigate the effects that the synthetic pathways exert on theentire metabolic network of a photosynthetic organism, a number of theproposed cycles in this example have been inserted into the metabolicnetwork reconstruction of Chlamydamonas rhinhartti. For each cycle, themodel is set up by removing Rubisco, adding the cycle, and running thesimulations under the same environmental conditions as published(aerobic conditions with ample light and CO₂), with few modifications:Ferredoxin located in the thylakoid lumen was moved into the chloroplaststroma, as supported by the literature. In addition, mitochondriallactate dehydrogenase (UQ containing), Isocitrate lyase, and carbonicanhydrase were changed from irreversible to reversible since noliterature evidence could be found to support the irreversibledesignation. Adenylate kinase was also added to the chloroplast since43% of this enzyme activity was localized to the chloroplast.

2. Flux Balance Analysis and the “Committing Reactions” of the CarbonFixation Pathways

Flux balance analysis (FBA) was used for determining a measure of thestoichiometric and topological efficiency of each cycle in the contextof the Chlamydomonas metabolic network. FBA is a useful tool to computegrowth yields and likely secretion products. For each cycle, FBA wasused to compute these quantities given a constraint on the “committingreaction”. A “committing reaction”, by definition, is the reaction fromthe pathway of interest which connects the carbon flux in the pathway tothe rest of the metabolic network. In each simulation, the committingreaction was constrained to a flux of 5 mmol carbon atoms per g DryWeight biomass per hour. To compare the efficiency of different cycles,the FBA results were used to compute a molar ratio of secretion productsto biomass production under a defined committing reaction flux (FIG. 12,Table 1). Thereby, this measure allows the comparison of how efficientlythe various cycles convert CO₂ into biomass.

The following reactions were used as committing reactions, followed bythe respective pathways:

-   Rubisco: Wild Type-   Glyoxylate Carboligase: MOG/lactate, MOG/alanine, KGS-ICDH, PyrS-ME,    PyrS-PEPC, AcC-ICDH/glycerate & PrC-KGS/Glutamate pathways.-   Citramalyl-CoA lyase: 3-HP & AcC-PrC-KGS/Glutamate pathways.-   Carboxytransferase: AcC-PrC/Citrate-   Acetoacetyl-CoA transporter: 3-HP/4-HB pathway    3. The Choice of Electron Acceptor has the Greatest Effect on the    Growth Rate

In silico, wild-type Chlamydomonas successfully assimilates CO₂ andconverts all of it to biomass. When Rubisco is removed and syntheticpathways are added, the parameter which affects biomass the most is theelectron carriers employed in each cycle. In general, cycles which useonly NADPH or ferredoxin are capable of the same growth yield as wildtype Chlamydomonas (FIG. 12, Table 1). The only exceptions are cases inwhich the end product of the cycle is acetyl-CoA (as discussed below).

Cycles which employ the electron carriers FAD or ubiquinone (UQ)consistently have a lower growth yield since the pathways needed torecycle the reduced ubiquinone pass the electrons onto acetyl-coa whichis converted into ethanol and secreted. A similar phenomenon occurs whenacetyl-CoA serves as the product of the carbon fixation cycle. This isbecause the metabolism of acetyl-CoA leads to an increased usage ofNAD⁺, thereby increasing the amount of NADH which must be recycled. Theprimary mechanisms which effectively replenish the pool of NAD⁺ areethanol production and oxidative phosphorylation. Indeed, in both casesmitochondrial ATP synthase carries a higher flux than needed for maximalgrowth. However, this still is unable to recycle all of the NADH;therefore, the other mechanism of ethanol production is needed tomaintain steady state amounts of NAD⁺/NADH. The molar ratios of secretedcarbon to fixed carbon, for the pathways analyzed, are given in FIG. 12,Table 1.

4. The Replacement of Rubisco does not Significantly Change the OverallCandidate Flux Distributions

It has been previously demonstrated that cells tend to minimize thechanges in flux vales following a genetic perturbation in the metabolicnetwork. This occurs because the genetic changes often require thedifferential expression of many enzymes which may or may not correspondto established regulatory patterns. If a large portion of the metabolicnetwork undergoes significant changes, this can inhibit growth until thestrain evolves and adapts to the new network topology. In more extremeconditions, such shifts in the metabolic flux distributions may inhibitthe acceptance of such pathways. Therefore, when replacing naturalmetabolic pathways, it is beneficial to identify synthetic pathwayswhich minimize the number of reactions that experience a significantchange in the range of allowable flux.

To evaluate the extent by which the allowable steady state fluxdistributions are affected by the replacement of Rubisco withalternative carbon fixation pathways, uniform random sampling of theallowable flux phenotypes is employed here. In this method, instead ofsearching for the optimal solution as done in FBA, the distribution ofallowable fluxes for all reactions are found for the entire solutionspace. However, since it is preferable to only look at the constraintson higher growth, only the space of phenotypes which maintain at least95% of the optimal growth yield as computed using FBA is analyzed here(this was repeated for 90% and 99% of the optimal growth yield; however,qualitative results did not change). For all reactions which wereconsistent between the two models, a p-value was computed for the nullhypothesis that the two distributions overlap. The number ofsignificantly different reactions was then determined using a FDR of0.05. The percents of fluxes that were changed significantly by theaddition of the synthetic pathways are given in FIG. 12, Table 1, foreach analyzed cycle.

Interestingly, the MOG pathways require the fewest number of reactionsto significantly change the allowable flux range (as low as 12-13%;FDR=0.05). On average, ferredoxin containing cycles witness slightlyhigher numbers of significantly changing reactions. Ubiquinione orFAD-containing cycles and cycles which produce acetyl-CoA, however,demonstrate significantly higher changes in the allowable fluxdistributions (FIG. 12, Table 1). Therefore we predict that NADPH orferredoxin containing pathways will be more likely to be accepted andrequire less time for adaptation to the new distribution of metabolitefluxes throughout the metabolic network.

5. Randomly Swapping NADPH to NADH Occasionally Affects the Growth Rate

Multiple enzymes may employ different electron donors at differentefficiencies, or have isoforms which can use different electron donors.Two commonly interchangeable electron donors are NADH and NADPH. Toprobe how varying the usage of these carriers affects the efficiency,reactions requiring such an electron carrier were randomly chosenthroughout the network. Electron carriers were changed from NADPH toNADH and the growth yield was computed. In the majority of the changesthere is little or no affect on the growth rate for the various cycles.However, a select few enzymes, when changed, affected growth in one ormore cycle. Those enzymes include:

-   Lactate dehydrogenase (MOG/lactate pathways);-   Succinate dehydrogenase (3-HP and 3-HP/4-HB pathways)-   Isocitrate dehydrogenase (3-HP/4-HB, PyrS-ME & PyrS-PEPC pathways);-   GASP dehydrogenase (phosphorylating) (PyrS-ME & PyrS-PEPC pathways);-   Malate dehydrogenase (PyrS-ME & PyrS-PEPC pathways);-   Malic enzyme (PyrS-ME pathway);-   Malonyl-CoA reductase (3-HP cycle);

This suggests that in most cases cycles are robust to changes in similarelectron carriers, but also that these few enzymes can be adjusted toproduce more biomass or secretion products as needed.

6. Conclusion

Combined, the results from analyzing a subset of synthetic cycles in thecontext of the Chlamydomonas metabolic network suggests thatglyoxylate-producing cycles employing NADPH as electron donors, such asthe MOG cycles, will have a growth yield comparable to network with therPP cycle. These cycles are topologically the most efficient, anddisrupt the allowable flux distributions the least. Combined with theprediction of improved kinetics in comparison to the rPP cycle, thesecycles demonstrate great promise for the optimization of CO₂sequestration and biomass production. Alternatively, the UQ-containingcycles may be used for the production of useful secretion products suchas biofuels.

TABLE 17 E.C. number Enzyme 1.1.1.28 Lactate Dehydrogenase 4.2.1.54Lactoyl-CoA Dehydratase 1.1.1.37/82 Malate Dehydrogenase (NAD/NADP)2.3.3.9 Malate Synthase 1.1.1.38/39/40 Malic enzyme 1.1.1.— +Malonyl-CoA Reductase 1.2.1.— (Hydroxypropionate-forming) 1.2.1.—Malonyl-CoA Reductase (Malonatesemialdehyde-forming) 4.1.3.24 Malyl-CoALyase 6.2.1.9 Malyl-CoA Synthetase ND Mesaconyl-CoA Hydratase 4.3.1.2Methylaspartate Ammonia-Lyase 6.4.1.4 Methylcrotonyl-CoA Carboxylase2.1.3.1 Methylmalonyl-CoA Carboxytransferase 5.1.99.1 Methylmalonyl-CoAEpimerase 5.4.99.2 Methylmalonyl-CoA Mutase 4.1.3.24 Methylmalyl-CoALyase ND Methylsuccinyl-CoA Dehydrogenase 6.2.1.8. Oxalate CoA Ligase3.7.1.1 Oxaloacetase 4.1.1.31 PEP Carboxylase 2.7.2.3 PhosphoglycerateKinase 5.4.2.1 Phosphoglycerate Mutase 2.7.1.19 Phosphoribulokinase2.8.3.1 Propionate CoA Transferase 6.4.1.3 Propionyl-CoA Carboxylase6.2.1.— + Propionyl-CoA Synthase 4.2.1.— + 1.3.1.— 6.4.1.1 PyruvateCarboxylase 1.2.7.1 Pyruvate Synthase 2.7.9.2 Pyruvate Water (Phosphate)Dikinases (2.7.9.1) 5.3.1.6 Ribose-5-Phosphate Isomerase 4.1.1.39Ribulose-Bisphosphate Carboxylase 5.1.3.1 Ribulose-Phosphate 3-Epimerase3.1.3.37 Sedoheptulose-Bisphosphatase 1.3.5.1/ SuccinateDehydrogenase/Fumarate Reductase 1.3.99.1/ 1.3.1.6 2.8.3.7Succinate-citramalate CoA-transferase ND Succinyl-CoA Reductase6.2.1.4/5 Succinyl-CoA Synthetase 1.1.1.60 Tartronate-SemialdehydeReductase 2.2.1.1 Transketolase 5.3.1.1 Triose-Phosphate Isomerase1.1.99.2 2-Hydroxyglutarate Dehydrogenase 2.3.3.11 2-HydroxyglutarateSynthase 4.2.1.— 2-Hydroxyglutaryl-CoA Dehydratase 6.4.1.72-Ketoglutarate Carboxylase 1.2.7.3 2-Ketoglutarate Synthase 4.2.1.342-Methylmalate Dehydratase 1.1.1.157 3-Hydroxybutyryl-CoA Dehydrogenase1.1.1.61 4-Hydroxybutyrate Dehydrogenase ND 4-Hydroxybutyryl-CoADehydratase ND 4-Hydroxybutyryl-CoA Synthetase 2.8.3.8 Acetate:SuccinateCoA-Transferase 2.3.1.9/16 Acetyl-CoA C-Acyltransferase 6.4.1.2Acetyl-CoA Carboxylase 6.2.1.13 Acetyl-CoA Synthetase 4.2.1.3 AconitateHydratase Alanine Aminomutase 4.3.1.1 Aspartate Ammonia-Lyase 2.6.1.1Aspartate Transaminase 2.3.3.8 ATP Citrate Lyase 2.6.1.18beta-Alanine-Pyruvate Transaminase 4.1.3.22 Citramalate Lyase 4.1.3.25Citramalyl-CoA Lyase 2.3.3.1 Citrate Synthase ND Crotonyl-CoACarboxylase/Reductase 4.2.1.11 Enolase 4.2.1.17/55 Enoyl-CoA Hydratase(Crotonase) ND Ethylmalonyl-CoA Epimerase ND Ethylmalonyl-CoA Mutase3.1.3.11 Fructose-Bisphosphatase 4.1.2.13 Fructose-Bisphosphate Aldolase4.2.1.2 Fumarate Hydratase 2.8.3.12 Glutaconate CoA-Transferase 1.4.1.4Glutamate Dehydrogenase (NADPH) 5.4.99.1 Glutamate Mutase 1.2.1.12/13Glyceraldehyde-3P Dehydrogenase (Phosphorylating) 2.7.1.31 GlycerateKinase 4.1.1.47 Glyoxylate Carboligase 1.2.1.17 Glyoxylate Dehydrogenase1.1.1.41/42 Isocitrate Dehydrogenase 4.1.3.1 Isocitrate Lyase

Example 3 Practical Implementation

In Vitro Implementation

The capture of inorganic carbon from the atmosphere by industrial meanshas received significant attention in recent years. Apart fromcompletely chemical approaches, the in vitro reconstitution of theCalvin-Benson Cycle was proposed and pursued as an efficient alternativeto perform this goal, while providing voluble bioorganic compounds. Theproposed synthetic carbon fixation pathways can achieve the same goalwith less enzymatic biomass. Tables 18 and 19 present the enzymaticconstituents of such an in vitro carbon fixation system, operating theC4-Glyoxylate/Alanine or C4-Glyoxylate/Lactate pathway, where for eachenzyme prokaryotic and eukaryotic alternatives are given, if possible.

To support carbon the enzymatic system should be provided with energizedcofactors, namely ATP and NADH and/or NADPH. These cofactors can beregenerated in vitro in various ways. Notably, if one would like toprovide reducing power using a single type of electron donor (NADPHonly) one should choose malate dehydrogenase from higher plants andlactate dehydrogenase from Trichomonas (see Table 18 and 19).

Bacterial Implementation, E. coli

Implementing the synthetic carbon fixation pathways in the naturallyheterotrophic E. coli might be extremely beneficial. Adapting thishighly utilized organism to an autotrophic way of life can open newroutes for its cultivation in the biotechnology industries and for theproduction of a large variety of voluble compounds.

Apart from an active carbon fixation pathway the organism will need asustainable source of energy and reducing power, in order to achieveautotrophy. In most cases through respiration, the source of reducingpower will also generate the required energy as long as oxygen isavailable. NAD⁺ is the preferred intermediate electron acceptor becauseit can directly serve both as an electron donor for carbon fixation andas an energy producer when oxidized by E. coli's respiratory electronchain. The two best candidates for providing E. coli with reducing power(and energy) are formate and phosphite. The soluble enzymeNAD⁺-dependent formate dehydrogenase irreversibly oxidizes formate(E′⁰=−430 mV, and reduces NAD⁺) (formate cannot be directly assimilatedby E. coli). The recently discovered enzyme NAD:phosphite oxidoreductaseirreversibly oxidizes phosphite to phosphate (E′⁰=−650 mV) and reducesNAD⁺. Both enzymes operate under fully aerobic conditions which enablemolecular oxygen to serve as the terminal electron acceptor, maximizingthe energetic gain of oxidizing the electron donors. Both are used toregenerate NAD(P) and both retain full activity in E. coli. Notably,both enzymes were evolved to accept and even prefer NADP over NAD.

A further option is to establish an E. coli strain that is capable ofgrowing using electrical power as the sole source of reducing power andenergy, where electrodes will supply the cells with electrons. Intact E.coli cells cannot directly react with an electrode, but redox dyes canmediate electron transfer from the electrode to the dye and then intocellular metabolism. An excellent example for this is the electronophoreneutral red, which was shown to reduce NAD in vivo in the gram-negativebacterium Actinobacillus succinogenes. Notably, the organism was shownto grow using reduced neutral red as the sole electron donor formetabolism.

E. coli endogenously operates the enzymes Pyruvate Dikinase, PEPCarboxylase, Malate Dehyderogenase, Lactate Dehydrogenase, GlyoxylateCarboligase, Tartronate-Semialdehyde Reductase and Glycerate Kinase.

To implement the C4-Glyoxylate/Alanine pathway in this organism thefollowing foreign enzymes should be expressed: Malyl-CoA Synthetase,Malyl-CoA Lyase, Methylmalonyl-CoA Carboxytransferase, MalonateSemialdehyde Dehydrogenase, Alanine Aminomutase and Beta-AlaninePyruvate Transaminase. The prokaryotic sources, as appear in Table 18,are more suitable to this host.

To implement the C4-Glyoxylate/Lactate pathway in this organism thefollowing foreign enzymes should be expressed: Malyl-CoA Synthetase,Malyl-CoA Lyase, Methlmalonyl-CoA Carboxytransferase, Malonyl-CoAReductase, Propionate CoA Transferase, Enoyl-CoA Hydratase andLactoyl-CoA dehydratase. The prokaryotic sources, as appear in Table 1,are more suitable to this host.

Cyanobacterial Implementation, Synechocystis sp. Strain PCC6803

The fresh water cyanobacterium Synechocystis sp. strain PCC6803 is anexcellent candidate for carbon fixation manipulation. First, being aprokaryote, it is relatively free of compartmentalization issues. Inaddition, it is easily transformed with foreign DNA. Most importantly,it can attain both autotrophic as well as heterotrophic mode of growth,depending on the availability of light. This metabolic versatility makesthis organism an ideal candidate for an extreme metabolic modification,which might become much more difficult in other cyanobacterial strainsthat can grow only autotrophically and cannot survive without theactivity of Rubisco.

Synechocystis sp. strain PCC6803 endogenously operates the enzymesPyruvate Dikinase, PEP Carboxylase, Malate Dehyderogenase, GlyoxylateCarboligase, Tartronate-Semialdehyde Reductase and Glycerate Kinase.

To implement the C4-Glyoxylate/Alanine pathway in this organism thefollowing foreign enzymes should be expressed: Malyl-CoA Synthetase,Malyl-CoA Lyase, Methlmalonyl-CoA Carboxytransferase, MalonateSemialdehyde Dehydrogenase, Alanine Aminomutase and Beta-AlaninePyruvate Transaminase. The prokaryotic sources, as appear in Table 18,are more suitable to this host.

To implement the C4-Glyoxylate/Lactate pathway in this organism thefollowing foreign enzymes should be expressed: Malyl-CoA Synthetase,Malyl-CoA Lyase, Methlmalonyl-CoA Carboxytransferase, Malonyl-CoAReductase, Propionate CoA Transferase, Enoyl-CoA Hydratase andLactoyl-CoA dehydratase. The prokaryotic sources, as appear in Table 18,are more suitable to this host. The enzyme Lactate Dehydrogenase wasfound to operate in the cyanobacterium Synechocystis but was not provedto exist in Synechocystis. Therefore, if this enzyme is indeed absent inSynechocystis, the Synechocystis Lactate Dehydrogenase should beexpressed.

If the cycles are to be operated using NADPH as the sole electron donor,malate dehydrogenase from higher plants and lactate dehydrogenase fromTrichomonas should be also expressed (see Table 18 and 19).

Algae Implementation, Chlamydomonas reinhardtii:

Chlamydomonas reinhardtii as one of the simplest unicellular, eukaryote,phototrophic organism; which make it a good candidate for carbonfixation modification. While the organism does posses the enzymes PEPCarboxylase, Malate Dehyderogenase and Glycerate Kinase, the former oneis not localized to the chloroplast. This will necessitate expressingthis endogenous gene with a particular chloroplast targeting signal.

To implement the C4-Glyoxylate/Alanine pathway in this organism thefollowing foreign enzymes should be expressed: Pyruvate Dikinase,Malyl-CoA Synthetase, Malyl-CoA Lyase, Methlmalonyl-CoACarboxytransferase, Malonate Semialdehyde Dehydrogenase, AlanineAminomutase, Beta-Alanine Pyruvate Transaminase, Glyoxylate Carboligaseand Tartronate-Semialdehyde Reductase. Various sources of these enzymesare given in Table 18. All these genes should be expressed with achloroplast targeting signal.

To implement the C4-Glyoxylate/Lactate pathway in this organism thefollowing foreign enzymes should be expressed: Pyruvate Dikinase,Malyl-CoA Synthetase, Malyl-CoA Lyase, Methlmalonyl-CoACarboxytransferase, Malonyl-CoA Reductase, Propionate CoA Transferase,Enoyl-CoA Hydratase, Lactoyl-CoA dehydratase, Lactate Dehydrogenase,Glyoxylate Carboligase and Tartronate-Semialdehyde Reductase. Varioussources of these enzymes are given in Table 18. All these genes shouldbe expressed with a chloroplast targeting signal.

C3-Plant Implementation, Tobacco (Nicotiana)

Tobacco is one of the most studied C3-plant. While the organism doesposses the enzymes Pyruvate Dikinase, PEP Carboxylase, MalateDehyderogenase and Glycerate Kinase, the former two are not localized tothe chloroplast. This will necessitate expressing these endogenous geneswith a particular chloroplast targeting signal.

To implement the C4-Glyoxylate/Alanine pathway in this organism thefollowing foreign enzymes should be expressed: Malyl-CoA Synthetase,Malyl-CoA Lyase, Methlmalonyl-CoA Carboxytransferase, MalonateSemialdehyde Dehydrogenase, Alanine Aminomutase, Beta-Alanine PyruvateTransaminase, Glyoxylate Carboligase and Tartronate-SemialdehydeReductase. Various sources of these enzymes are given in table 18. Allthese genes should be expressed with a chloroplast targeting signal.

To implement the C4-Glyoxylate/Lactate pathway in this organism thefollowing foreign enzymes should be expressed: Malyl-CoA Synthetase,Malyl-CoA Lyase, Methlmalonyl-CoA Carboxytransferase, Malonyl-CoAReductase, Propionate CoA Transferase, Enoyl-CoA Hydratase, Lactoyl-CoAdehydratase, Lactate Dehydrogenase, Glyoxylate Carboligase andTartronate-Semialdehyde Reductase. Various sources of these enzymes aregiven in Table 18. All these genes should be expressed with achloroplast targeting signal.

TABLE 18 The C4-Glyoxylate/Alanine pathway Eukaryotic Prokayotic ECOrigin Origin number Enzyme Maize, E. coli (20, 21) 2.7.9.1/2 PyruvateDikinase chloroplast (24, Synechocystis 25) (22, 23) Tobacco (26) Maize,E. coli (27, 28) 4.1.1.31 PEP Carboxylase chloroplast (30) SynechococcusTobacco (31) (29) Chlamydomonas Synechocystis (32) (22, 23) E. coli (33)1.1.1.37 NAD Malate Synechocystis Dependent Dehyderogenase (22)Trichomonas ‡ (34) Maize, 1.1.1.82 NADP chloroplast (35) DependentTobacco (36) Chlamydomonas (37) Pseudomonas 6.2.1.9 Malyl-CoA Synthetase(38, 39) Pseudomonas 4.1.3.24 Malyl-CoA Lyase (40) Wheat germsPropionibacterium 2.1.3.1 Methylmalonyl-CoA (43, 44) (41, 42)Carboxytransferase Metallosphaera (45) — Malonate SemialdehydeSulfolobus (45) Dehydrogenase Bacillus † (46, 47) — Alanine AminomutaseClostridium †† (48, 49) Wax bean, Pseudomonas (50) 2.6.1.18 Beta-AlaninePyruvate cotyledons (52) Bacillus (51) Transaminase E. coli (53)4.1.1.47 Glyoxylate Carboligase Synechocystis (54) E. coli (55) 1.1.1.60Tartronate-Semialdehyde Synechocystis Reductase (54) Maize, E. coli (56)2.7.1.31 Glycerate Kinase chloroplast (57) Synechocystis Arabidopsis,(54) chloroplast (58) Chlamydomonas, chloroplast (58) † Evolved fromBacillus subtilis lysine aminomutase †† Clostridium subterminale lysineaminomutase can catalyze also the alanine amniomutase reaction, oxygensensitive ‡ A bifunctional enzyme, catalyzing both malate and lactateoxidation.

TABLE 19 The C4-Glyoxylate/Lactate pathway Eukaryotic Prokayotic ECOrigin Origin number Enzyme Maize, E. coli (20, 21) 2.7.9.1/2 PyruvateDikinase chloroplast (24, Synechocystis 25) (22, 23) Tobacco (26) Maize,E. coli (27, 28) 4.1.1.31 PEP Carboxylase chloroplast (30) SynechococcusTobacco (31) (29) Chlamydomonas Synechocystis (32) (22, 23) E. coli (33)1.1.1.37 NAD Malate Synechocystis Dependent Dehyderogenase (22)Trichomonas ‡ (34) Maize, 1.1.1.82 NADP chloroplast (35) DependentTobacco (36) Chlamydomonas (37) Pseudomonas 6.2.1.9 Malyl-CoA Synthetase(38, 39) Pseudomonas 4.1.3.24 Malyl-CoA Lyase (40) Wheat germsPropionibacterium 2.1.3.1 Methlmalonyl-CoA (43, 44) (41, 42)Carboxytransferase Chloroflexus (59) — Malonyl-CoA Reductase Clostridium(60, 2.8.3.1 Propionate CoA Transferase 61) E. coli (62, 63) 4.2.1.17/55Enoyl-CoA Hydratase Pigeon (65) Clostridium † (64) 4.2.1.54 Lactoyl-CoAdehydratase Pseudomonas (65) E. coli (66) 1.1.1.28 NAD LactateSynechococcus Dependent Dehydrogenase (67) Trichomonas ‡ (34) Bacillus†† (68) NADP Dependent E. coli (53) 4.1.1.47 Glyoxylate CarboligaseSynechocystis (54) E. coli (55) 1.1.1.60 Tartronate-SemialdehydeSynechocystis Reductase (54) Maize, E. coli (56) 2.7.1.31 GlycerateKinase chloroplast (57) Synechocystis Arabidopsis, (54) chloroplast (58)Chlamydomonas, chloroplast (58) † Oxygen sensitive †† Accepts both NADand NADP ‡ A bifunctional enzyme, catalyzing both malate and lactateoxidation.

REFERENCES

-   1. Chakrabarti S, Bhattacharya S, & Bhattacharya S K (2003)    Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase:    a step toward carbon dioxide fixation bioprocess. Biotechnol Bioeng    81(6):705-711.-   2. Bhattacharya S, Schiavone M, Gomes J, & Bhattacharya S K (2004)    Cascade of bioreactors in series for conversion of    3-phospho-D-glycerate into D-ribulose-1,5-bisphosphate: kinetic    parameters of enzymes and operation variables. J Biotechnol    111(2):203-217.-   3. Mahato S, et al. (2004) Potential use of sugar binding proteins    in reactors for regeneration of CO2 fixation acceptor    D-Ribulose-1,5-bisphosphate. Microb Cell Fact 3(1):7.-   4. Wichmann R & Vasic-Racki D (2005) Cofactor Regeneration at the    Lab Scale. Technology Transfer in Biotechnology, Advances in    Biochemical Engineering/Biotechnology, (Springer Berlin/Heidelberg),    Vol 92, pp 225-260.-   5. Unden G & Bongaerts J (1997) Alternative respiratory pathways of    Escherichia coli: energetics and transcriptional regulation in    response to electron acceptors. Biochim Biophys Acta    1320(3):217-234.-   6. Thauer R K, Jungermann K, & Decker K (1977) Energy conservation    in chemotrophic anaerobic bacteria. Bacteriol Rev 41(1): 100-180.-   7. Friedebold J & Bowien B (1993) Physiological and biochemical    characterization of the soluble formate dehydrogenase, a    molybdoenzyme from Alcaligenes eutrophus. J Bacteriol    175(15):4719-4728.-   8. Costas A M, White A K, & Metcalf W W (2001) Purification and    characterization of a novel phosphorus-oxidizing enzyme from    Pseudomonas stutzeri WM88. J Biol Chem 276(20):17429-17436.-   9. Vrtis J M, White A K, Metcalf W W, & van der Donk W A (2001)    Phosphite dehydrogenase: an unusual phosphoryl transfer reaction. J    Am Chem Soc 123(11):2672-2673.-   10. van der Donk W A & Zhao H (2003) Recent developments in pyridine    nucleotide regeneration. Curr Opin Biotechnol 14(4):421-426.-   11. Gul-Karaguler N, Sessions R B, Clarke A R, & Holbrook J J (2001)    A single mutation in the NAD-specific formate dehydrogenase from    Candida methylica allows the enzyme to use NADP Biotechnology    Letters 23(4):283-287.-   12. Serov A E, Popova A S, Fedorchuk V V, & Tishkov V I (2002)    Engineering of coenzyme specificity of formate dehydrogenase from    Saccharomyces cerevisiae. Biochem J 367(Pt 3):841-847.-   13. Tishkov V I & Popov V O (2006) Protein engineering of formate    dehydrogenase. Biomol Eng 23(2-3):89-110.-   14. Rissom S, Schwarz-Linek U, Vogel M, Tishkov V I, & Kragl    U (1997) Synthesis of chiral var epsilon-lactones in a two-enzyme    system of cyclohexanone mono-oxygenase and formate dehydrogenase    with integrated bubble-free aeration Tetrahedron: Asymmetry    8(15):2523-2526.-   15. Woodyer R, van der Donk W A, & Zhao H (2003) Relaxing the    nicotinamide cofactor specificity of phosphite dehydrogenase by    rational design. Biochemistry 42(40):11604-11614.-   16. Park D H & Zeikus J G (1999) Utilization of electrically reduced    neutral red by Actinobacillus succinogenes: physiological function    of neutral red in membrane-driven fumarate reduction and energy    conservation. J Bacteriol 181(8):2403-2410.-   17. Park D H, Laivenieks M, Guettler M V, Jain M K, & Zeikus J    G (1999) Microbial utilization of electrically reduced neutral red    as the sole electron donor for growth and metabolite production.    Appl Environ Microbiol 65(7):2912-2917.-   18. Anderson S L & McIntosh L (1991) Light-activated heterotrophic    growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a    blue-light-requiring process. J Bacteriol 173(9):2761-2767.-   19. Peltier J B, et al. (2000) Proteomics of the chloroplast:    systematic identification and targeting analysis of lumenal and    peripheral thylakoid proteins. Plant Cell 12(3):319-341.-   20. Narindrasorasak S & Bridger W A (1977) Phosphoenolypyruvate    synthetase of Escherichia coli: molecular weight, subunit    composition, and identification of phosphohistidine in phosphoenzyme    intermediate. J Biol Chem 252(10):3121-3127.-   21. Berman K M & Cohn M (1970) Phosphoenolpyruvate synthetase of    Escherichia coli. Purification, some properties, and the role of    divalent metal ions. J Biol Chem 245(20):5309-5318.-   22. CyanoBase (Synechocystis sp. PCC 6803, GeneView.).-   23. Kaneko T, et al. (1996) Sequence analysis of the genome of the    unicellular cyanobacterium Synechocystis sp. strain PCC6803. II.    Sequence determination of the entire genome and assignment of    potential protein-coding regions. DNA Res 3(3):109-136.-   24. Chastain C J, et al. (2000) Further analysis of maize C(4)    pyruvate, orthophosphate dikinase phosphorylation by its    bifunctional regulatory protein using selective substitutions of the    regulatory Thr-456 and catalytic His-458 residues. Arch Biochem    Biophys 375(1):165-170.-   25. Sugiyama T (1973) Purification, molecular, and catalytic    properties of pyruvate phosphate dikinase from the maize leaf.    Biochemistry 12(15):2862-2868.-   26. Aoyagi K & Bassham J A (1984) Pyruvate Orthophosphate Dikinase    of C3 Seeds and Leaves as Compared to the Enzyme from Maize. Plant    Physiol 75:387-392.-   27. Kai Y, et al. (1999) Three-dimensional structure of    phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric    inhibition. Proc Natl Acad Sci USA 96(3):823-828.-   28. Yano M & Izui K (1997) The replacement of Lys620 by serine    desensitizes Escherichia coli phosphoenolpyruvate carboxylase to the    effects of the feedback inhibitors L-aspartate and L-malate. Eur J    Biochem 247(1):74-81.-   29. Chen L M, Omiya T, Hata S, & Izui K (2002) Molecular    characterization of a phosphoenolpyruvate carboxylase from a    thermophilic cyanobacterium, Synechococcus vulcanus with unusual    allosteric properties. Plant Cell Physiol 43(2): 159-169.-   30. Uedan K & Sugiyama T (1976) Purification and Characterization of    Phosphoenolpyruvate Carboxylase from Maize Leaves. Plant Physiol    57(6):906-910.-   31. Koizumi N, Sato F, & Yamada Y (1996) Bacterial production and    purification of phosphorylatable phosphoenolpyruvate carboxylase    from tobacco. Biosci Biotechnol Biochem 60(12):2089-2091.-   32. Mamedov T G, Moellering E R, & Chollet R (2005) Identification    and expression analysis of two inorganic C- and N-responsive genes    encoding novel and distinct molecular forms of eukaryotic    phosphoenolpyruvate carboxylase in the green microalga Chlamydomonas    reinhardtii. Plant J 42(6):832-843.-   33. Park J S, et al. (2007) Escherichia coli malate dehydrogenase, a    novel solubility enhancer for heterologous proteins synthesized in    Escherichia coli. Biotechnol Lett 29(10):1513-1518.-   34. Wu G, Fiser A, ter Kuile B, Sali A, & Muller M (1999) Convergent    evolution of Trichomonas vaginalis lactate dehydrogenase from malate    dehydrogenase. Proc Natl Acad Sci USA 96(11):6285-6290.-   35. Kagawa T & Bruno P L (1988) NADP-malate dehydrogenase from    leaves of Zea mays: purification and physical, chemical, and kinetic    properties. Arch Biochem Biophys 260(2):674-695.-   36. Backhausen J E & Scheiber R (1999) Adaptation of tobacco plants    to elevated CO2: influence of leaf age on changes in physiology,    redox states and NADP-malate dehydrogenase activity. Journal of    experimental botany 50(334):665-675.-   37. Lemaire S D, et al. (2005) NADP-malate dehydrogenase from    unicellular green alga Chlamydomonas reinhardtii. A first step    toward redox regulation? Plant Physiol 137(2):514-521.-   38. Hersh L B (1974) Malate thiokinase. The reaction mechanism as    determined by initial rate studies. J Biol Chem 249(19):6264-6271.-   39. Elwell M & Hersh L B (1979) Substrate-dependent dissociation of    malate thiokinase. J Biol Chem 254(7):2434-2438.-   40. Hacking A J & Quayle J R (1974) Purification and properties of    malyl-coenzyme A lyase from Pseudomonas AM1. Biochem J    139(2):399-405.-   41. Shenoy B C, Xie Y, Sha D, & Samols D (1993) Identification and    characterization of a factor which is essential for assembly of    transcarboxylase. Biochemistry 32(40): 10750-10756.-   42. Xie Y, Shenoy B C, Magner W J, Hejlik D P, & Samols D (1993)    Purification and characterization of the recombinant 5 S subunit of    transcarboxylase from Escherichia coli. Protein Expr Purif    4(5):456-464.-   43. Hatch M D & Stumpf P K (1961) Fat metabolism in higher    plants. XVI. Acetyl coenzyme A carboxylase and acyl coenzyme    A-malonyl coenzyme A transcarboxylase from wheat germ. J Biol Chem    236:2879-2885.-   44. Hatch M D & Stumpf P K (1962) Fat Metabolism in Higher    Plants. XVII. Metabolism of Malonic Acid & Its alpha-Substituted    Derivatives in Plants. Plant Physiol 37(2):121-126.-   45. Alber B, et al. (2006) Malonyl-coenzyme A reductase in the    modified 3-hydroxypropionate cycle for autotrophic carbon fixation    in archaeal Metallosphaera and Sulfolobus spp. J Bacteriol    188(24):8551-8559.-   46. Chen D, Ruzicka F J, & Frey P A (2000) A novel lysine    2,3-aminomutase encoded by the yodO gene of bacillus subtilis:    characterization and the observation of organic radical    intermediates. Biochem J 348 Pt 3:539-549.-   47. Liao H H, Gokarn R R, Gort S J, Jessen H J, & Selifonova    O (2007) U.S. Pat. No. 7,309,597 B2.-   48. Frey P A & Ruzicka F J (2003).-   49. Ruzicka F J, Lieder K W, & Frey P A (2000) Lysine    2,3-aminomutase from Clostridium subterminale SB4: mass spectral    characterization of cyanogen bromide-treated peptides and cloning,    sequencing, and expression of the gene kamA in Escherichia coli. J    Bacteriol 182(2):469-476.-   50. C. U. I, et al. (2006) One-pot synthesis of amino-alcohols using    a de-novo transketolase and beta-alanine:pyruvate transaminase    pathway in Escherichia coli. Biotechnol. Bioeng. 96:559-569.-   51. Nakano Y, Tokunaga H, & Kitaoka S (1977) Two omega-amino acid    transaminases from Bacillus cereus. J Biochem 81(5):1375-1381.-   52. Stinson R A & Spencer M S (1969) Beta alanine    aminotransferase (s) from a plant source. Biochem Biophys Res Commun    34(1):120-127.-   53. Chang Y Y, Wang A Y, & Cronan J E, Jr. (1993) Molecular cloning,    DNA sequencing, and biochemical analyses of Escherichia coli    glyoxylate carboligase. An enzyme of the acetohydroxy acid    synthase-pyruvate oxidase family. J Biol Chem 268(6):3911-3919.-   54. Eisenhut M, et al. (2006) The plant-like C2 glycolate cycle and    the bacterial-like glycerate pathway cooperate in phosphoglycolate    metabolism in cyanobacteria. Plant Physiol 142(1):333-342.-   55. Njau R K, Herndon C A, & Hawes J W (2000) Novel beta-hydroxyacid    dehydrogenases in Escherichia coli and Haemophilus influenzae. J    Biol Chem 275(49):38780-38786.-   56. Doughty C C & Hayashi J A (1975) D-glycerate 3-kinase from    Escherichia coli. Methods Enzymol 42:124-127.-   57. Kleczkowski L A & Randall D D (1988) Purification and    characterization of D-glycerate 3-kinase from maize leaves. Planta    173:221-229.-   58. Boldt R, et al. (2005) D-GLYCERATE 3-KINASE, the last unknown    enzyme in the photorespiratory cycle in Arabidopsis, belongs to a    novel kinase family. Plant Cell 17(8):2413-2420.-   59. Hugler M, Menendez C, Schagger H, & Fuchs G (2002)    Malonyl-coenzyme A reductase from Chloroflexus aurantiacus, a key    enzyme of the 3-hydroxypropionate cycle for autotrophic CO(2)    fixation. J Bacteriol 184(9):2404-2410.-   60. Schweiger G & Buckel W (1984) On the dehydration of (R)-lactate    in the fermentation of alanine to propionate by Clostridium    propionicum. FEBS Lett 171(1):79-84.-   61. Valentin H E & Steinbuchel A (1994) Application of enzymatically    synthesized short-chain-length hydroxy fatty acid coenzyme A    thioesters for assay of polyhydroxyalkanoic acid synthases. Applied    Microbiology and Biotechnology 40(5):699-709.-   62. Yang S Y, Li J M, He X Y, Cosloy S D, & Schulz H (1988) Evidence    that the fadB gene of the fadAB operon of Escherichia coli encodes    3-hydroxyacyl-coenzyme A (CoA) epimerase, delta 3-cis-delta    2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to    3-hydroxyacyl-CoA dehydrogenase. J Bacteriol 170(6):2543-2548.-   63. Agnihotri G & Liu H W (2003) Enoyl-CoA hydratase. reaction,    mechanism, and inhibition. Bioorg Med Chem 11(1):9-20.-   64. Hofmeister A E & Buckel W (1992) (R)-lactyl-CoA dehydratase from    Clostridium propionicum. Stereochemistry of the dehydration of    (R)-2-hydroxybutyryl-CoA to crotonyl-CoA. Eur J Biochem    206(2):547-552.-   65. Vagelos P R, Earl J M, & Stadtman E R (1959) Propionic acid    metabolism. II. Enzymatic synthesis of lactyl pantethine. J Biol    Chem 234(4):765-769.-   66. Dym O, Pratt E A, Ho C, & Eisenberg D (2000) The crystal    structure of D-lactate dehydrogenase, a peripheral membrane    respiratory enzyme. Proc Natl Acad Sci USA 97(17):9413-9418.-   67. Sanchez J J, Palleroni N J, & Doudoroff M (1975) Lactate    dehydrogenases in cyanobacteria. Arch Microbiol 104(1):57-65.-   68. Romero S, Merino E, Bolivar F, Gosset G, & Martinez A (2007)    Metabolic engineering of Bacillus subtilis for ethanol production:    lactate dehydrogenase plays a key role in fermentative metabolism.    Appl Environ Microbiol 73(16):5190-5198.

Example 4 Constructing an Autotrophic Strain of E. coli as a ModelOrganism to Test the Synthetic Carbon Fixation Pathways

In order to test and compare the proposed synthetic carbon fixationcycles in vivo, there is a need to find a suitable autotrophic host thatis capable of growth when supplied with inorganic carbon, a source ofenergy and reducing power (electrons). The present inventors willutilize the well-studied model organism, E. coli, and adapt it to anautotrophic mode of growth using the classic Calvin-Benson Cycle. NativeE. coli contains most of the Calvin-Benson Cycle enzymes as part of thepentose phosphate pathway and the gluconeogenesis pathway. In fact, theonly two enzymes missing to support a full operational cycle are PRK(phosphorubilokinase) and Rubisco (FIG. 15). These two enzymes from atleast two origins, the cyanobacterium Synechococcus and thepurple-bacterium Rhodospirillum rubrum, were successfully expressed inE. Coli and showed activity significant enough to affect the host'sgrowth. The Type II Rubisco from R. rubrum might be a preferable choicebecause it can fold correctly in the host even with the low levels ofchaperones in the E. coli cells. As a further alternative, the presentinventors will express an operon of the proteobacteria Ralstoniaeutropha, which contains all the Calvin-Benson Cycle genes in tandem. Toconfirm the full operation of the cycle in the host, the presentinventors will feed the transformed E. coli with ¹³CO₂ and follow the¹³C signal across the cycle using liquid-chromatography massspectrometry (LC-MS) measurements. If the cycle operates as a whole onecan expect to find ¹³C enrichment in triose-phosphates,hexose-phosphates and pentose-phosphates. By using available standardlibraries of metabolites, one can identify, with the highest precision,the compounds of interest in the LC-MS output and quantify theirconcentrations in the host.

To provide E. coli with the necessary energy and reducing power(electrons) needed for growth either the enzyme NAD⁺-dependent formatedehydrogenase or the enzyme NAD⁺-dependent phosphite dehydrogenase canbe used. Both enzymes catalyze irreversible reactions (formate→CO₂ orphosphite→phosphate) and both operate under fully aerobic conditionswhich enable molecular oxygen to serve as the terminal electronacceptor. Both are used to regenerate NAD(P) and are known to retainedfull activity in E. coli.

After the host is proven to operate a functioning carbon fixation cycleand to have a constant energy supply it can be forced to grow using onlyinorganic carbon. Two parallel approaches may be used: (A) Transferringthe cells from a media containing a carbon source to a carbon-freemedia; (B) Decreasing the carbon source concentration gradually until itbecomes negligible.

Establishing an operative Calvin-Benson Cycle may be performed both byexpressing single enzymes from different origins and by expressing wholeoperons from foreign sources as detailed above. In addition, the energyand reducing power could be supplied using at-least two parallelsystems. As an essential debugging procedure LC-MS measurements may beused to track the flow of carbon in the metabolic network of the host.

The bacteria may be grown under autotrophic conditions for manygenerations and the adaptation process of the organism to these novelconditions may be tracked. LC-MS may be used to decipher themetabolomics and metabolite fluxes in the host. Feeding E. coli with¹³CO₂ will allow tracking of the roots by which carbon dioxide isassimilated in the bacteria.

Following establishment of an autotrophic strain of E. coli, thisorganism may be used to test and compare one of the proposed syntheticcarbon fixation cycles (described in Examples 1, 2 and 3) in vivo.Foreign enzymes will be expressed so that the host would be able tooperate an alternative carbon fixation cycle and then the Calvin-BensonCycle will be stopped by eliminating Rubisco and/or PRK. It would beextremely important to choose foreign enzymes from organisms withsimilar cellular conditions as in E. coli, if possible. These conditionsinclude pH, temperature, ionic strength and a prokaryotic environment asopposed to eukaryotic one.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A system for carbon fixation, comprising anelectron donor and enzymes which catalyze reactions of a carbon fixationpathway, wherein all the carboxylation reactions of the carbon fixationpathway utilize: (i) phophoenolpyruvate (PEP) carboxylase and acetyl-CoAcarboxylase; or (ii) phophoenolpyruvate (PEP) carboxylase; or (iii)pyruvate carboxylase and acetyl-CoA carboxylase; or (iv) pyruvatecarboxylase wherein products of said reactions of the carbon fixationpathway comprise oxaloacetate and malonyl-CoA, and wherein an additionalproduct of the carbon fixation pathway is glyoxylate, wherein anin-organic carbon is introduced into a substrate to become a carboxylicacid group during said carboylation reactions, wherein said system forcarbon fixation is in a non-cellular particle.
 2. The system of claim 1,wherein said glyoxylate is an export product.
 3. The system of claim 1,wherein an export product of the carbon fixation pathway is pyruvate. 4.The system of claim 1, wherein the enzymes of the carbon fixationpathway generate more than 0.3 μmol glyceraldehyde-3-phosphate/min/mg.5. The system of claim 1, wherein said enzyme which performs saidcarboxylation enzyme is PEP carboxylase.
 6. The system of claim 1,wherein at least two of said reactions of the carbon fixation pathwayare carboxylation reactions.
 7. The system of claim 1, wherein one ofsaid reactions of the carbon fixation pathway utilizes methylmalonyl-CoAcarboxytransferase.
 8. The system of claim 1, wherein products of thereactions of the carbon fixation pathway further comprise pyruvate,phophoenolpyruvate (PEP), malate, malyl CoA and acetyl CoA.
 9. Thesystem of claim 1, wherein said electron donor is selected from thegroup consisting of ATP, NADH and NADPH.
 10. The system of claim 1wherein said non-cellular particle is selected from the group consistingof polymeric particles, microcapsules liposomes, microspheres,microemulsions, nano-plates, nanoparticles, nanocapsules andnanospheres.
 11. The system of claim 1, wherein the enzymes of saidcarboxylation reactions of the carbon fixation pathway consist of: (i)PEP carboxylase and acetyl-CoA carboxylase; or (ii) PEP carboxylase; or(iii) pyruvate carboxylase and acetyl-CoA carboxylase; or (iv) pyruvatecarboxylase.